Cancer vaccines

Abstract

The invention relates to combinations for treating or preventing cancer comprising a closed linear DNA molecule and an mRNA molecule. The invention also relates to a closed linear DNA molecule and an mRNA molecule for use in a heterologous prime-boost immunization regimen in a subject. In addition, the invention relates to methods and kits incorporating and / or using the closed linear DNA molecule and the mRNA molecule.

Description

[0001] CANCER VACCINES

[0002] TECHNICAL FIELD

[0003] The invention relates to combinations for treating or preventing cancer comprising a closed linear DNA molecule and an mRNA molecule. The invention also relates to a closed linear DNA molecule and an mRNA molecule for use in a heterologous prime-boost immunization regimen in a subject. In addition, the invention relates to methods and kits incorporating and / or using the closed linear DNA molecule and the mRNA molecule.

[0004] BACKGROUND

[0005] Genetic vaccines, including mRNA and DNA based platforms, have emerged as versatile tools in gene therapy, viral vaccine development, and cancer vaccine research. These vectors offer a platform for delivering genetic material encoding specific antigens, thereby eliciting targeted immune responses against viral pathogens or tumor cells. However, despite their widespread use, a significant challenge remains in tailoring these approaches to individual patients due to the inherent heterogeneity of tumors and immune responses.

[0006] Traditionally, the "one vector fits all" approach has been applied in the development of viral vaccines and cancer vaccines targeting shared antigens or tumor-associated antigens (TAAs). While this strategy has shown promise in eliciting broad immune responses against viral antigens, it may fall short in the context of personalized cancer vaccines, where the antigens are unique to each patient's tumor. In light of this limitation, there is a growing recognition of the need for more customizable vaccine platforms that can accommodate the diverse antigenic profiles of individual tumors. Thus, a need exists for an effective vaccine platform for treating or preventing cancer in an individual patient.

[0007] An effective cancer vaccine should follow the successful model of viral vaccine, requiring the selection of specific antigens for each cancer type or individual tumor. This is challenging because cancer cells originate from host cells and have primarily "self antigens, which are subject to central and peripheral tolerance, meaning they lack cognate antigen-specific T cells in the host’s immune system. For an effective cancer vaccine, the selected antigen must be: 1) cancer-specific to avoid targeting healthy tissues, 2)"non-self to ensure the presence of functional antigen-specific T cells that can expand upon antigen delivery, 3) immunodominant to maximize the immune response against cancer cells. Identifying these optimal cancer antigens quickly and on a large scale is a challenge. Tumors consist of genetically heterogeneous collections of cells with varying antigen repertoires, similar to pathogen serotypes. These antigens can evolve over time due to immune pressure, further complicating the selection process. In summary, cancer antigens must be selected to optimize four key vaccine characteristics: specificity (ensure the vaccine targets cancer cells specifically, sparing healthy tissues), immunogenicity: (choose antigens that elicit a strong immune response), valency (cover all possible cancer clones and subclones), and durability (maintain long-term protection by adapting to changes in the cancer cell population. Finally, optimal vaccination platforms need to deliver antigens quickly, effectively, and safely.

[0008] Prime-boost vaccination strategies involve multiple immunizations. They aim to improve the effectiveness of the vaccine. Generally, the same vaccine composition is administered each time - a so-called homologous prime-boost vaccination regimen. Homologous vaccination refers to an immunization regimen using the same vaccine for both the prime (first immunization) and boost (second or any further immunization). On the other hand, heterologous prime-boost immunization requires that a different vaccine is used as the primary and for at least some of the later immunizations. Heterologous prime-boost vaccination strategies have been tested for the development of vaccines against infectious diseases such as HIV, SIV, HCV, HSV and HBV and are generally based on combinations of DNA and viral vaccines (Kardani, K., Bolhassani, A. & Shahbazi, S. Prime-boost vaccine strategy against viral infections: Mechanisms and benefits. Vaccine 34, 413-423 (2016)).

[0009] DESCRIPTION

[0010] The invention provides a combination comprising a closed linear DNA molecule and an mRNA molecule, wherein the closed linear DNA molecule and / or the mRNA molecule is / are comprised in a nanoparticle. The combination may be a kit. That is to say that the closed linear DNA molecule and the mRNA molecule may be comprised in a kit. The inventors have shown that the combination of a closed linear DNA molecule and an mRNA molecule in the context of the heterologous prime-boost immunization regiment is advantageous over a homologous vaccination regiment.

[0011] The combination (or kit) is particularly suitable for use in personalized medicine, for example, to treat or prevent cancer in a subject. Thus, the invention provides a closed linear DNA molecule encoding at least one neoantigen, and an mRNA molecule encoding at least one neoantigen, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0012] The closed linear DNA molecule and / or the mRNA molecule may be comprised in a vaccine composition. Thus, the invention provides a combination (or kit) comprising: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen; and b. a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen; wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0013] The compositions of the first and second vaccine composition are interchangeable. All features, examples and embodiments described herein in the context of the first vaccine composition comprising a closed linear DNA molecule and the second vaccine composition comprising an mRNA molecule apply mutatis mutandis to the combination comprising a first vaccine composition comprising an mRNA molecule and a second vaccine composition comprising a closed linear DNA molecule.

[0014] The closed linear DNA molecule may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 neoantigens.

[0015] The mRNA molecule may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 neoantigens.

[0016] The closed linear DNA molecule and the mRNA molecule may encode the same neoantigens or different neoantigen(s). Preferably, the closed linear DNA molecule and the mRNA molecule encode the same neoantigen(s). For example, the closed linear DNA molecule and the mRNA molecule may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 of the same neoantigens.

[0017] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens; wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0018] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least twenty neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least twenty neoantigens; wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle. The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least forty neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least forty neoantigens; wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0019] Although different combinations of the closed linear DNA molecule and the mRNA molecule were tested and showed positive results, the inventors found that a heterologous prime / boost system in which the closed linear DNA molecule is delivered by electroporation, followed by an mRNA molecule delivered by encapsulation in a nanoparticle shows a particularly good level of immune response as well as increased survival of animal subjects suffering from cancer. This particular prime / boost regimen offers further benefits in terms of efficient manufacture. Specifically, the closed linear DNA molecule requires no formulation, a key benefit compared to the longer process needed for the mRNA formulation in a nanoparticle. This is because patients primed with a closed linear DNA molecule (via electroporation) can receive a boost 3 weeks later, which corresponds to the time needed for the preparation of the mRNA- nanoparticle formulation. Thus, preferably, the mRNA molecule is comprised in a nanoparticle. Preferably, the closed linear DNA molecule is not comprised in a nanoparticle. Thus, the combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen; and b. a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen, wherein the mRNA molecule is comprised in a nanoparticle.

[0020] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens, wherein the mRNA molecule is comprised in a nanoparticle.

[0021] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least twenty neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least twenty neoantigens, wherein the mRNA molecule is comprised in a nanoparticle.

[0022] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least forty neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least forty neoantigens, wherein the mRNA molecule is comprised in a nanoparticle.

[0023] The combination may further comprise a third vaccine composition. The third vaccine composition may comprise an mRNA molecule or a closed linear DNA molecule. Preferably, the third vaccine composition comprises an mRNA molecule. The mRNA molecule of the third vaccine composition may encode at least one neoantigen. The mRNA molecule of the third vaccine composition may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 neoantigens. The mRNA molecule of the third vaccine composition may be the same as the mRNA molecule of the second vaccine composition. The third vaccine composition may be the same as the second vaccine composition.

[0024] The combination may further comprise a fourth (or subsequent, i.e. a fifth, sixth, etc.) vaccine composition. The fourth (or subsequent) vaccine composition may comprise an mRNA molecule or a closed linear DNA molecule. The mRNA molecule or the closed linear DNA molecule of the fourth (or subsequent) vaccine composition may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 neoantigens.

[0025] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen; and b. a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen; c. a third vaccine composition comprising an mRNA molecule encoding at least one neoantigen; wherein the closed linear DNA molecule and / or the mRNA molecule(s) are comprised in a nanoparticle.

[0026] The combination may further comprise an immune checkpoint inhibitor.

[0027] The nanoparticle may comprise: a. a lipid component, wherein the lipid component is one or more ionizable lipids and / or one or more cationic lipids; b. a phospholipid; c. a steroid lipid; and d. a cationic polymer.

[0028] The invention also provides a combination or kit-of-parts comprising (a) a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen; and (b) a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen, for simultaneous, separate or sequential use in treating or preventing cancer in a subject.

[0029] The invention also provides a combination or kit-of-parts of (a) a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen; and (b) a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen, for simultaneous, separate or sequential use in treating or preventing cancer in a subject.

[0030] As used herein, the “first” and “second” (and any subsequent) numbers to define the vaccine compositions are not intended to specify the order of administration or delivery, unless specifically stated, especially in the context of administration regimens. Instead, the terms “first” and “second” are intended to describe “different” vaccine compositions.

[0031] Preferably, the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0032] A combination or kit-of-parts comprising (a) a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens; and (b) a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens, may be for simultaneous, separate or sequential use in treating or preventing cancer in a subject, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0033] The invention also provides a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen for use in treating or preventing cancer, wherein the first vaccine composition is administered to a subject simultaneously, separately or sequentially with a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0034] A first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens may be used in treating or preventing cancer, wherein the first vaccine composition is administered to a subject simultaneously, separately or sequentially with a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0035] The invention also provides a second vaccine composition comprising a mRNA molecule encoding at least one neoantigen for use in treating or preventing cancer, wherein the second vaccine composition is administered to a subject simultaneously, separately or sequentially with a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0036] A second vaccine composition comprising a mRNA molecule encoding at least two neoantigens may be used in treating or preventing cancer, wherein the second vaccine composition is administered to a subject simultaneously, separately or sequentially with a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0037] The invention also provides a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen for use in a heterologous prime-boost immunization regimen in a subject, wherein the first vaccine composition is administered at least once to the subject, and wherein a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen is separately administered to the subject following at least one administration of the first vaccine, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0038] A first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens may be used in a heterologous prime-boost immunization regimen in a subject, wherein the first vaccine composition is administered at least once to the subject, and wherein a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens is separately administered to the subject following at least one administration of the first vaccine, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0039] The invention also provides a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen for use in treating or preventing cancer in a subject, wherein the first vaccine composition is administered at least once to the subject, and wherein a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen is separately administered to the subject following at least one administration of the first vaccine, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0040] A first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens may be used in treating or preventing cancer in a subject, wherein the first vaccine composition is administered at least once to the subject, and wherein a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens is separately administered to the subject following at least one administration of the first vaccine, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0041] The invention also provides a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen for use in a heterologous prime-boost immunization regimen in a subject, wherein the second vaccine composition is administered to the subject following at least one administration of a first vaccine composition, and wherein the first vaccine composition comprises a closed linear DNA molecule encoding at least one neoantigen, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0042] A second vaccine composition comprising an mRNA molecule encoding at least two neoantigens may be used in a heterologous prime-boost immunization regimen in a subject, wherein the second vaccine composition is administered to the subject following at least one administration of a first vaccine composition, and wherein the first vaccine composition comprises a closed linear DNA molecule encoding at least two neoantigens, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0043] The invention also provides a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen for use in treating or preventing cancer in a subject, wherein the second vaccine composition is administered to the subject following at least one administration of a first vaccine composition, and wherein the first vaccine composition comprises a closed linear DNA molecule encoding at least one neoantigen, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0044] A second vaccine composition comprising an mRNA molecule encoding at least two neoantigens may be used in treating or preventing cancer in a subject, wherein the second vaccine composition is administered to the subject following at least one administration of a first vaccine composition, and wherein the first vaccine composition comprises a closed linear DNA molecule encoding at least two neoantigens, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0045] It is to be understand that, in some embodiments, the second vaccine composition comprising an mRNA molecule encoding at least one (preferably at least two) neoantigens is administered to a subject before the first vaccine composition comprising a closed linear DNA molecule encoding at least one (preferably at least two) neoantigens.

[0046] Thus, the invention provides a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen for use in a heterologous prime-boost immunization regimen in a subject, wherein the second vaccine composition is administered at least once to the subject, and wherein a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen is separately administered to the subject following at least one administration of the second vaccine, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0047] The invention also provides a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen for use in treating or preventing cancer in a subject, wherein the second vaccine composition is administered at least once to the subject, and wherein a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen is separately administered to the subject following at least one administration of the second vaccine, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0048] The description of the embodiments in which the first vaccine composition is administered to the subject first, before the administration of the second vaccine composition apply mutatis mutandis to the embodiments in which the second vaccine composition is administered to the subject first, before the administration of the first vaccine composition. Thus, the skilled person would understand that the first vaccine composition and the second vaccine composition can be administered to the subject in any order.

[0049] The invention also provides a closed linear DNA molecule as described herein, wherein the closed linear DNA molecule encodes at least 40 neoantigens.

[0050] The invention also provides an mRNA molecule as described herein, wherein the mRNA molecule encodes at least 40 neoantigens.

[0051] The invention provides a composition comprising a closed linear DNA molecule as described herein, wherein the closed linear DNA molecule encodes at least 40 neoantigen, wherein the closed linear DNA molecule is encapsulated in a nanoparticle as described herein.

[0052] The invention provides a composition comprising an mRNA molecule as described herein, wherein the mRNA molecule encodes at least 40 neoantigen, wherein the mRNA molecule is encapsulated in a nanoparticle as described herein.

[0053] The mRNA molecule, the closed linear DNA molecule and the nanoparticle are described in detail in the sections below. It would be understood that the description provided herein in the context of vaccine compositions applies more generally to the mRNA molecules of the invention and the closed linear DNA molecules of the invention.

[0054] Each aspect or embodiment as defined herein may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0055] Unless otherwise defined herein, scientific, and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition.

[0056] It should be understood that singular prepositions such as “a,” “an,” and “the,” are often used for convenience, however, all instances of the singular are intended to encompass the plural unless otherwise indicated either explicitly or from context. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Further, it should be understood that all references, including journal articles, books, patents, technical documents, and the like, mentioned in this disclosure are hereby incorporated by reference in their entirety and for all purposes.

[0057] Vaccine compositions

[0058] The invention provides a combination comprising: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen; and b. a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen; wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0059] The term “vaccine composition” as used herein is intended to encompass any compositions which are suitable for use as vaccines, i.e. to elicit or maintain an immune response in a subject. The vaccine composition may include only one (i.e. a single) ingredient, e.g. either a closed linear DNA molecule or an mRNA molecule as described herein. In a prime / boost system, typically, “a first vaccine composition” is used as a primer to elicit an initial immune response in a subject, whereas “a second vaccine composition” is used as booster to elicit secondary immune responses, or to maintain the initial immune response elicited by the primer. The first vaccine composition may comprise a closed linear DNA molecule or an mRNA molecule. The second vaccine composition may comprise a closed linear DNA molecule or an mRNA molecule. The “third” (or subsequent) vaccine composition is also typically used as a booster. However, embodiments in which the “second vaccine composition” is administered before the “first vaccine composition” are also envisaged.

[0060] The vaccine composition may include additional components, for example, an adjuvant, a lipid, a peptide, or components of a nanoparticle. The vaccine composition may include a buffer, a carrier or an excipient. The vaccine composition may comprise a DNA molecule expressing IL-12 and / or IL-2. The vaccine composition may comprise an mRNA molecule expressing IL-12 and / or IL-2.

[0061] The closed linear DNA molecule as described herein or the mRNA molecule as described herein may be formulated (e.g. in a vaccine composition) as pills, tablets or capsules combined with one or more pharmaceutically acceptable solid carriers or as a solution in one or more pharmaceutically acceptable solvents, or as an emulsion, suspension or dispersion in one or more pharmaceutically acceptable solvents or carriers. The vaccine composition may also include other pharmaceutically acceptable excipients such as stabilizers, anti-oxidants, binders, colouring agents or emulsifying or taste-modifying agents and extended release formulations.

[0062] The vaccine composition may be administered orally, topically, parenterally or transdermally or by inhalation. The vaccine composition may be administered by injection or intravenous infusion using suitable sterile solutions. Topical dosage forms may be creams, ointments, patches, or similar vehicles suitable for transdermal and topical dosage forms.

[0063] The vaccine composition may be dissolved or suspended in a liquid vehicle or formulated as a granule (a small particle or grain), a pellet (a small sterile solid mass consisting of a highly purified composition, with or without excipients, made by the formation of granules, or by compression and moulding), or a pellet coated extended release (a solid dosage form in which the composition itself is in the form of granules to which varying amounts of coating have been applied, and which releases the composition in such a manner to allow a reduction in dosing frequency as compared to that composition presented as a conventional dosage form).

[0064] Other forms of vaccine composition include pills (a small, round solid dosage form containing the composition intended for oral administration), powder (an intimate mixture of dry, finely divided composition with one or more pharmaceutically acceptable additives that may be intended for internal or external use), elixir (a clear, pleasantly flavoured, sweetened hydroalcoholic liquid containing dissolved composition; it is intended for oral use), chewing gum (a sweetened and flavoured insoluble plastic material of various shapes which when chewed, releases the composition into the oral cavity), syrup (an oral solution containing the composition and high concentrations of sucrose or other sugars; the term has also been used to include any other liquid dosage form prepared in a sweet and viscid vehicle, including oral suspensions), tablet (a solid dosage form containing the composition with or without suitable diluents), tablet chewable (a solid dosage form containing the composition with or without suitable diluents that is intended to be chewed, producing a pleasant tasting residue in the oral cavity that is easily swallowed and does not leave a bitter or unpleasant after-taste), tablet coated or tablet delayed release, tablet dispersible, tablet effervescent, tablet extended release, tablet film coated, or tablet film coated extended release where the tablet is formulated in such manner as to make the contained composition available over an extended period of time following ingestion. In other forms of vaccine composition, a tablet for solution, tablet for suspension, tablet multilayer, tablet multilayer extended release may be provided, where the tablet is formulated in such manner as to allow at least a reduction in dosing frequency as compared to that composition presented as a conventional dosage form. A tablet orally disintegrating, tablet orally disintegrating delayed release, tablet soluble, tablet sugar coated, osmotic, and the like are also suitable.

[0065] The oral dosage form vaccine composition may contain, in addition to the composition, one or more inactive pharmaceutical ingredients such as diluents, solubilizers, alcohols, binders, controlled release polymers, enteric polymers, disintegrants, excipients, colorants, flavorants, sweeteners, antioxidants, preservatives, pigments, additives, fillers, suspension agents, surfactants (e.g., anionic, cationic, amphoteric and nonionic), and the like. Various FDA-approved topical inactive ingredients are found at the FDA's “The Inactive Ingredients Database” that contains inactive ingredients specifically intended as such by the manufacturer.

[0066] As used herein, injectable and infusion dosage forms include, but are not limited to, a liposomal injectable, which either consists of or forms liposomes (a lipid bilayer vesicle usually composed of phospholipids which is used to encapsulate the composition); an injection, which includes a sterile preparation intended for parenteral use; an emulsion injection, which includes an emulsion consisting of a sterile, pyrogen-free preparation intended to be administered parenterally; or a lipid complex injection.

[0067] For example, the vaccine composition can be administered by intratympanic injection (e.g. into the middle ear) and / or injections into the outer, middle, and / or inner ear. Such methods are routinely used in the art, for example, for the administration of steroids and antibiotics into human ears. Injection can be, for example, through the round window of the ear or through the cochiear capsule.

[0068] Other forms of vaccine composition include a powder for solution injection, which is a sterile preparation intended for reconstitution to form a solution for parenteral use; a powder for suspension injection that is a sterile preparation intended for reconstitution to form a suspension for parenteral use; a powder lyophilized for liposomal suspension injection, which is a sterile freeze dried preparation intended for reconstitution for parenteral use which has been formulated in a manner that would allow liposomes (a lipid bilayer vesicle usually composed of phospholipids which is used to encapsulate the composition, either within a lipid bilayer or in an aqueous space) to be formed upon reconstitution; or a powder lyophilized for solution injection, wherein lyophilization (“freeze drying”) is a process which involves the removal of water from products in the frozen state at extremely low pressures.

[0069] A suspension injection comprises a liquid preparation, suitable for injection, which consists of solid particles dispersed throughout a liquid phase in which the particles are not soluble that can also consist of an oil phase dispersed throughout an aqueous phase, or vice-versa. A suspension liposomal injection comprises a liquid preparation, suitable for injection, which consists of an oil phase dispersed throughout an aqueous phase in such a manner that liposomes (a lipid bilayer vesicle usually composed of phospholipids which is used to encapsulate the composition, either within a lipid bilayer or in an aqueous space) are formed. A suspension sonicated injection comprises a liquid preparation, suitable for injection, which consists of solid particles dispersed throughout a liquid phase in which the particles are not soluble. In addition, the product is sonicated while a gas is bubbled through the suspension, and this results in the formation of microspheres by the solid particles.

[0070] In another mode of administration, the vaccine composition can be administered in situ, via a catheter or pump. A catheter or pump can, for example, direct the composition into the target location.

[0071] The parenteral carrier system may include one or more pharmaceutically suitable excipients, such as solvents and co-solvents, solubilizing agents, wetting agents, suspending agents, thickening agents, emulsifying agents, chelating agents, buffers, pH adjusters, antioxidants, reducing agents, antimicrobial preservatives, bulking agents, protectants, tonicity adjusters, and special additives. Formulations suitable for parenteral administration conveniently comprise a sterile oily or aqueous preparation of the composition which is preferably isotonic with the blood of the recipient but this is not essential.

[0072] As used herein, inhalation dosage forms include, but are not limited to, an aerosol (a product that is packaged under pressure and contains the composition which is released upon activation of an appropriate valve system intended for topical application to the skin as well as local application into the nose (nasal aerosols), mouth (lingual and sublingual aerosols), or lungs (inhalation aerosols)). A foam aerosol is a dosage form containing the composition, surfactants, aqueous or nonaqueous liquids, and propellants, whereby if the propellant is in the internal (discontinuous) phase (i.e., of the oil-in-water type), a stable foam is discharged, and if the propellant is in the external (continuous) phase (i.e., of the water-in-oil type), a spray or a quick-breaking foam is discharged. A metered aerosol is a pressurized dosage form consisting of metered dose valves which allow for the delivery of a uniform quantity of spray upon each activation. A powder aerosol is a product that is packaged under pressure and contains the composition, in the form of a powder that is released upon activation of an appropriate valve system. An aerosol spray is an aerosol product which utilizes a compressed gas as the propellant to provide the force necessary to expel the product as a wet spray and being applicable to solutions of the composition in aqueous solvent(s).

[0073] A transdermal dosage form may include, but is not limited to, a patch (a drug delivery system that often contains an adhesive backing that is usually applied to an external site on the body, whereby the ingredients (including the composition) either passively diffuse from, or are actively transported from, some portion of the patch, and whereby depending upon the patch, the ingredients (including the composition are either delivered to the outer surface of the body or into the body. Various types of transdermal patches such as matrix, reservoir and others are known in the art.

[0074] A topical dosage form may include various dosage forms known in the art such as lotions (an emulsion, liquid dosage form, whereby this dosage form is generally for external application to the skin), lotion augmented (a lotion dosage form that enhances composition delivery, whereby augmentation does not refer to the strength of the composition in the dosage form), gels (a semisolid dosage form that contains a gelling composition to provide stiffness to a solution or a colloidal dispersion, whereby the gel may contain suspended particles) and ointments (a semisolid dosage form, usually containing less than 20% water and volatiles and greater than 50% hydrocarbons, waxes, or polyols as the vehicle, whereby this dosage form is generally for external application to the skin or mucous membranes). Further embodiments include ointment augmented (an ointment dosage form that enhances composition delivery, whereby augmentation does not refer to the strength of the composition in the dosage form), creams (an emulsion, semisolid dosage form, usually containing greater than 20% water and volatiles and / or less than 50% hydrocarbons, waxes, or polyols may also be used as the vehicle, whereby this dosage form is generally for external application to the skin or mucous membranes) and cream augmented (a cream dosage form that enhances composition delivery, whereby augmentation does not refer to the strength of the composition in the dosage form). As used herein, an “emulsion” refers to a dosage form consisting of a two-phase system comprised of at least two immiscible liquids, one of which is dispersed as droplets, internal or dispersed phase, within the other liquid, external or continuous phase, generally stabilized with one or more emulsifying agents, whereby emulsion is used as a dosage form term unless a more specific term is applicable (e.g. cream, lotion, ointment). Further embodiments include suspensions (a liquid dosage form that contains solid particles dispersed in a liquid vehicle), suspension extended release, pastes (a semisolid dosage form, containing a large proportion, 20-50%, of solids finely dispersed in a fatty vehicle, whereby this dosage form is generally for external application to the skin or mucous membranes), solutions (a clear, homogeneous liquid dosage form that contains one or more chemical substances dissolved in a solvent or mixture of mutually miscible solvents), and powders.

[0075] The topical dosage form composition contains the composition and one or more inactive pharmaceutical ingredients such as excipients, colorants, pigments, additives, fillers, emollients, surfactants (e.g., anionic, cationic, amphoteric and nonionic), penetration enhancers (e.g., alcohols, fatty alcohols, fatty acids, fatty acid esters and polyols), and the like. Various FDA-approved topical inactive ingredients are found at the FDA's “The Inactive Ingredients Database” that contains inactive ingredients specifically intended as such by the manufacturer.

[0076] The closed linear DNA molecule may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 neoantigens. Preferably, the closed linear DNA molecule encodes at least 20 neoantigens, for example, at least 40 neoantigens.

[0077] The closed linear DNA molecule may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 different neoantigens. Preferably, the closed linear DNA molecule encodes at least 20 different neoantigens, for example, at least 40 different neoantigens.

[0078] The mRNA molecule may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 neoantigens. Preferably, the mRNA molecule encodes at least 20 neoantigens, for example, at least 40 neoantigens.

[0079] The mRNA molecule may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 different neoantigens. Preferably, the mRNA molecule encodes at least 20 different neoantigens, for example, at least 40 different neoantigens.

[0080] The presence of a high number (e.g. 20 or 40) of different neoantigens accounts for heterogenicity of cancer cells. This is particularly useful in the context of prophylactic cancer vaccines for which the genetic mark up of cancer calls might not be known. The use of a higher number of neoantigens also allows for the generation of a more universal therapeutic cancer vaccine, which will be applicable to a higher proportion of patients suffering from a given cancer type.

[0081] The closed linear DNA molecule and the mRNA molecule may encode the same neoantigens or different neoantigen(s). Preferably, the closed linear DNA molecule and the mRNA molecule encode the same neoantigen(s). That is to say that, for example, the closed linear DNA molecule may encode 20 different neoantigens and the mRNA molecule may encode the same 20 different neoantigens. For example, the closed linear DNA molecule and the mRNA molecule may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 of the same neoantigens.

[0082] The closed linear DNA molecule may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 neoantigens recognized and / or presented by MHC class I. The closed linear DNA molecule may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 neoantigens recognized and / or presented by MHC class II. The closed linear DNA molecule may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 neoantigens recognized and / or presented by MHC class I and at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 neoantigens recognized and / or presented by MHC class II.

[0083] The mRNA molecule may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 neoantigens recognized and / or presented by MHC class I. The mRNA molecule may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 neoantigens recognized and / or presented by MHC class II. The mRNA molecule may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 neoantigens recognized and / or presented by MHC class I and at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 neoantigens recognized and / or presented by MHC class II.

[0084] Preferably, all neoantigens encoded by the nucleic acid molecule (i.e. the closed linear DNA molecule or the mRNA molecule) of the first vaccine composition and / or the second vaccine composition are encoded by a single type of nucleic acid molecule. That is to say that the first vaccine composition and / or the second vaccine composition (and any subsequent vaccine compositions) may only comprise a single type of the nucleic acid molecule encoding neoantigens. For example, the first vaccine composition may comprise a single type of the closed linear DNA molecule encoding at least 1 , at least 2, or at least 20 neoantigen(s). For example, the second vaccine composition may comprise a single type of the mRNA molecule encoding at least 1 , at least 2, or at least 20 neoantigen(s). The term “a single type” as used herein is intended to encompass nucleic acid molecules encoding the same neoantigens. For example, two mRNA molecules encoding at least 20 of the same neoantigens in the same order would be classified as the nucleic acid molecules “of the same type”. However, a first mRNA molecule encoding 2 neoantigens and a second mRNA molecule encoding 2 different neoantigens would not be classified as “the same type”. Thus, preferably, the first vaccine composition and / or the second vaccine composition comprise nucleic acid molecules which encode the same number of neoantigens and the same type of neoantigens. In other words, the first vaccine composition and / or the second vaccine composition my comprise an isomorphic (or homotypic) population of nucleic acid molecules.

[0085] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens; wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0086] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least twenty neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least twenty neoantigens; wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle. The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least forty neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least forty neoantigens; wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0087] Preferably, the mRNA molecule is comprised in a nanoparticle. Preferably, the closed linear DNA molecule is not comprised in a nanoparticle. Thus, the combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen; and b. a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen, wherein the mRNA molecule is comprised in a nanoparticle.

[0088] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens, wherein the mRNA molecule is comprised in a nanoparticle.

[0089] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least twenty neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least twenty neoantigens, wherein the mRNA molecule is comprised in a nanoparticle.

[0090] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least forty neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least forty neoantigens, wherein the mRNA molecule is comprised in a nanoparticle.

[0091] The combination may further comprise a third vaccine composition The third vaccine composition may comprise an mRNA molecule or a closed linear DNA molecule. Preferably, the third vaccine composition comprises an mRNA molecule. The mRNA molecule of the third vaccine composition may encode at least one neoantigen. The mRNA molecule of the third vaccine composition may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 neoantigens. The mRNA molecule of the third vaccine composition may be the same as the mRNA molecule of the second vaccine composition. The third vaccine composition may be the same as the second vaccine composition.

[0092] The combination may further comprise a fourth (or subsequent, i.e. a fifth, sixth, etc.) vaccine composition. The fourth (or subsequent) vaccine composition may comprise an mRNA molecule or a closed linear DNA molecule. The mRNA molecule or the closed linear DNA molecule of the fourth (or subsequent) vaccine composition may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 neoantigens.

[0093] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens, wherein the first vaccine composition is suitable for delivery by electroporation; and b. a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens, wherein the mRNA molecule is comprised in a nanoparticle.

[0094] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen; and b. a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen; c. a third vaccine composition comprising an mRNA molecule encoding at least one neoantigen; wherein the closed linear DNA molecule and / or the mRNA molecule(s) are comprised in a nanoparticle.

[0095] Preferably, the second vaccine comprising an mRNA molecule and the third vaccine composition comprising an mRNA molecule are both comprised in a nanoparticle. Preferably, the first vaccine composition comprising a closed linear DNA molecule is not comprised in a nanoparticle. Instead, the first vaccine composition may be delivered to a subject by electroporation.

[0096] The combination may further comprise an immune checkpoint inhibitor. The immune checkpoint inhibitor may be administered to a subject before or after the first vaccine composition is administered. The immune checkpoint inhibitor may be administered to a subject before or after the second vaccine composition is administered. Preferably, the immune checkpoint inhibitor is administered to the subject before the first vaccine and the second vaccine compositions are administered. The immune checkpoint inhibitor may be an anti-CTLA-4 molecule, an anti-PD-1 molecule, an anti-PD-L1 molecule, an anti-LAG-3 molecule. Preferably, the immune checkpoint inhibitor is an anti-CTLA-4 molecule.

[0097] The closed linear DNA molecule may encode at least one neoantigen. The closed linear DNA molecule may comprise a double-stranded DNA portion that is closed at a first end by a first singlestranded portion (i.e. it may comprise a first hairpin at the first end) and closed at a second end by a second single-stranded portion (i.e. it may comprise a second hairpin at the second end). The closed DNA molecule may be a covalently-closed linear DNA molecule. The covalently-closed linear DNA molecule may comprise a first adaptor molecule at a first end and a second adaptor molecule at a second end. The first adaptor molecule and the second adaptor molecule may each comprise a hairpin. The hairpin may confer resistance to nuclease (e.g. exonuclease) digestion. The closed linear DNA molecule may comprise one or more protected nucleotides (i.e. nucleotides resistant to nuclease (e.g. exonuclease) digestion). The closed linear DNA molecule may be (i) a DNA molecule processed with TelN protelomerase; or (ii) a DNA molecule having a double-stranded portion closed at a first end by ligation of a first adaptor to the first end and closed at a second end by the ligation of a second adaptor to the second end.

[0098] Preferably, the closed linear DNA molecule is not incorporated into the genome of an animal (e.g. mouse or human) it is administered to.

[0099] The closed linear DNA molecules (defined as “closed linear DNA products”) and the methods for preparation thereof are described in W02023 / 006978 A1 , which is incorporated herein in its entirety.

[0100] Briefly, the closed linear DNA molecule may be produced by a method comprising:

[0101] (a) contacting a double-stranded DNA molecule with an endonuclease and first and second adaptor molecules to form a single contiguous aqueous volume; and

[0102] (b) incubating the single contiguous aqueous volume to generate the closed linear DNA molecule, wherein the closed linear DNA molecule comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein the linear double-stranded region is closed at a first end by the first adaptor molecule and closed at a second end by the second adaptor molecule.

[0103] The step of contacting the double-stranded DNA molecule with the endonuclease and first and second adaptor molecules is preferably performed in the presence of a ligase.

[0104] The closed linear DNA molecules may be partially double-stranded and / or partially single-stranded. The closed linear DNA molecules may comprise a portion that is double-stranded and a portion that is single-stranded. The closed linear DNA molecule may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding at least one neoantigen. Thus, the cassette may may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 neoantigens. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosomal binding site and a translational termination sequence. The cassette may additionally comprise sequences aiding protein expression, such as a cap-independent translation element. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, poly C, polyT or polyG sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the cassette, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0105] The sequences encoding neoantigen(s) may be separated by at least one linker. For example, each sequence of a neoantigen may be separated by at least one linker. Thus, the closed linear DNA molecule may encode at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 linkers. The linker may comprise amino acids AAY or GS. The linker may be AAY or GSGSGSGSGS.

[0106] The closed linear DNA molecule may comprise a spacer. The spacer may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs long. The closed linear DNA molecule may comprise an Inverted Terminal Repeat (ITR) sequence. However, preferably, the closed linear DNA molecule does not comprise an Inverted Terminal Repeat (ITR) sequence.

[0107] The closed linear DNA molecule may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11 ,000, at least 12,000, at least 13,000, at least 14,000, at least 15,000, at least 20,000, at least 25,000, or at least 30,000 base pairs long. Preferably, the closed linear DNA molecule is at least 500 base pairs long.

[0108] The linear double-stranded region (e.g. the linear portion of the double-stranded molecule) may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11 ,000, at least 12,000, at least 13,000, at least 14,000, at least 15,000, at least 20,000, at least 25,000, or at least 30,000 base pairs long. Preferably, the linear double-stranded region is at least 500 base pairs long.

[0109] The first adaptor molecule and / or the second adaptor molecule may be a synthetic adaptor molecule.

[0110] The first adaptor molecule may be a nucleic acid adaptor molecule. The second adaptor molecule may be a nucleic acid adaptor molecule. The first adaptor molecule and / or the second adaptor molecule may comprise a self-complementary element which creates a loop, such as a hairpin loop or a stem loop. Thus, the first adaptor molecule may comprise a hairpin or a stem-loop. The second adaptor molecule may comprise a hairpin or a stem-loop. Both the first and second adaptor molecules may comprise a hairpin or a stem-loop. The adaptor molecules may each comprise a double-stranded portion comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are linked together in a hairpin such that the sense strand is hybridized to the antisense strand. The double-stranded portion of an adaptor may comprise a 3’ overhang or a 5’ overhang of at least 1 , at least 2, at least 3, at least 4, or at least 5 nucleotides. Preferably the 3’ overhang or the 5’ overhang is 4-8 nucleotides. Each end of the linear double-stranded region (or linear portion of the double-stranded DNA molecule) may comprise a 3’ or a 5’ overhang. A portion of the first adaptor molecule (e.g. the overhang) may be complementary to the first end of the linear double-stranded region. A portion of the second adaptor molecule may be complementary to the second end of the linear double-stranded region.

[0111] The closed linear DNA molecule may be a covalently closed linear DNA molecule. Thus, the adaptor molecules close the ends of the linear double-stranded region forming a covalently closed linear DNA molecule. The first adaptor molecule and / or the second adaptor molecule may comprise a single-stranded portion. The single-stranded portion may form a hairpin or a stem-loop. Thus, the first adaptor molecule and / or the second adaptor molecule may comprise a loop portion. The single-stranded portion may comprise less than 10, 9, 8, 7, 6, 5, 4, 3, 2 nucleotides. The single-stranded portion may comprise at least 10, at least 15, at least 20, or at least 25 nucleotides.

[0112] The first adaptor molecule and / or the second adaptor molecule may comprise a double-stranded portion. The double-stranded portion may comprise less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, or less than 10 base pairs. The doublestranded portion may comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, or at least 15 base pairs.

[0113] The first adaptor molecule may comprise a portion that is complementary to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second adaptor molecule may comprise a portion that is complementary to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The first adaptor molecule may comprise a portion that anneals to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second adaptor molecule may comprise a portion that anneals to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The first adaptor molecule may comprise a portion that is complementary and anneals to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second adaptor molecule may comprise a portion that is complementary and anneals to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule).

[0114] The first adaptor molecule and / or the second adaptor molecule may comprise one or more protected nucleotides (i.e. nuclease-resistant nucleotides), such as phosphorothioated nucleotides. The protected nucleotides may be located in the single-stranded portion (e.g. hairpin portion) or the double-stranded portion. The protected nucleotides may be located in the overhang portion of the adaptor molecules.

[0115] The closed linear DNA molecule may comprise a plurality of phosphorothioated nucleotides at internal positions in each strand. For example, the closed linear DNA molecule may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. The internal positions may not be located between the second and penultimate nucleotide of the closed linear DNA molecule. The nucleotides resistant to exonuclease digestion (i.e. protected nucleotides) may be phosphorothioated nucleotides. For example, phosphorothioated nucleotides may be a-S-dATP (i.e. 2’-deoxyadenosine-5’-(a-thio)-triphosphate), a-S-dCTP (i.e. 2’-deoxycytidine-5’-(a-thio)-triphosphate), a-S-dGTP (i.e. 2’-deoxyguanosine-5’-(a-thio)-triphosphate), a-S-dTTP (i.e. 2’-deoxythymidine-5’-(a- thio)-triphosphate), a-S-dUTP (i.e. 2’-deoxyuridine-5’-(a-thio)-triphosphate), and / or uridine 2’, 3’- cyclophosphorothioate. The nucleotides resistant to exonuclease digestion (i.e. protected nucleotides) may be 2'-O-methyl nucleotides or 2'-O-methoxyethyl (MOE) nucleotides. For example, the MOE nucleotides may be 2’-O-methoxy-ethyl guanosine, 2’-O-methoxy-ethyl cytidine, 2’-O-methoxy-ethyl adenosine, and / or 2’-O-methoxy-ethyl thymidine.

[0116] The first adaptor molecule and / or the second adaptor molecule may comprise a functional portion. The functional portion may be a binding molecule, a targeting sequence, or a probe.

[0117] The mRNA molecule may comprise at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 150, at least 200 nucleotides, at least 500 nucleotides, at least 1 ,000 nucleotides, at least 2,000 nucleotides, at least 5,000 nucleotides, at least 10,000 nucleotides, at least 15,000 nucleotides, at least 16,000 nucleotides at least 20,000 nucleotides, at least 25,000 nucleotides, or at least 30,000 nucleotides. The mRNA molecule may comprise 5-30,000, 5-20,000, 6-19,000, 7-18,000, 8-17,000, 9-16,000, IQ- 15, 000, 10-13,000, 15-10,000, 20-5,000, 20-1 ,000, 20-500, or 25-400 nucleotides.

[0118] The mRNA molecule may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding at least one neoantigen. Thus, the cassette may may encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 neoantigens. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence.

[0119] The sequences encoding neoantigen(s) may be separated by at least one linker. For example, each sequence of a neoantigen may be separated by at least one linker. Thus, the mRNA molecule may encode at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 linkers, may comprise amino acids AAY or GS. The linker may be AAY or GSGSGSGSGS.

[0120] 24

[0121] 17044091 OMB OMB The mRNA molecule may comprise one or more protected nucleotides (i.e. nucleotides resistant to nuclease (e.g. exonuclease) digestion) (e.g. phosphorothioated nucleotides) as described herein.

[0122] The term “neoantigen” is intended to encompass a peptide that forms on cancer cells when certain mutations occur in tumour DNA. Cancer cells accumulate many DNA mutations that can alter the structure of proteins, which result in the formation of neoantigens. Therefore, neoantigens are a class of tumour-specific antigens that are absent from normal (i.e. healthy) tissue. The resulting neoantigens are displayed by human leukocyte antigens (HLA) on the surface of cancer cells, helping to stimulate immune responses when immune cells - such as T cells - recognize the neoantigens as “non-self.

[0123] The term “Major Histocompatibility Complexes” (MHC) (class I and II) refers to proteins found on the surfaces of cells that help the immune system recognize foreign substances. MHC proteins are found in all higher vertebrates. In human beings the complex is also called the human leukocyte antigen (HLA) system. There are two major types of MHC protein molecules — class I and class II. Class I MHC molecules span the membrane of almost every cell in an organism, while class II molecules are generally restricted macrophages and lymphocytes.

[0124] Neoantigens which can be encoded by the closed linear DNA molecule and / or the mRNA molecule as described herein will vary depending on the cancer type and the specific cancer mutations present in an individual subject (i.e. the patient). In the preventative context, the neoantigens may include the most popular neoantigens observed for a given cancer type. For example, for glioblastoma, the at least one neoantigen may be IDH1 . For example, for solid tumours, the at least one neoantigen may be Kras and / or P53.

[0125] In the context of a colorectal cancer, the at least one neoantigen may be resulting from a mutation in at least one of the gene(s): Wbp7- Kmt2a, Huwel , Hnrnpl, Latsl , Atg9a, Ndfip2, Slc35e4, Dpagtl , Nle1 , Hacel , Zbtb40, Pop1 , Actrl b, Tmem35, Adgpk, Repsi , pam, Gtf2i, and / or Spire"! . In the context of a colorectal cancer, the at least one neoantigen may be resulting from a mutation in at least one of the gene(s): Gpc1 , Tmem87, Slc20a1 , Als2, Tripl 2, Slc20a1 , Ackr3, Aldh18a1 , Ppp6r1 , Mtchl , Dhx35, Steap2, Dkk2, Gludl , Nphp3, Phykpl, Deptor, Drosha, Tdg, and / or E2f8.

[0126] The at least one neoantigen may be the neoantigen of any one of SEQ ID NOs: 2-41 .

[0127] In the context of a breast cancer, the at least one neoantigen may be resulting from a mutation in at least one of the gene(s): Ftsj3, Mastl, Mastl, Utp3, Brd1 , Phactr4, Bhlhe40, Proserl , Ktn1 , Bazla, Plekhg3, and / or Ric1 .

[0128] The at least one neoantigen may be the neoantigen of any one of SEQ ID NOs: 42-155.

[0129] 25

[0130] 17044091 OMB OMB In the therapeutic context, the neoantigens expressed by cancer cells of the individual subject (i.e. the patient) may be first established (e.g. by collecting a sample from a subject followed by sequencing) in order to determine to most suitable neoantigens for incorporation into the vaccine compositions as described herein. Alternatively, a vaccine encoding a high number of different neoantigens (e.g. at least 20 or at least 40) may be prepared and administered to account for tumour heterogen icity without the need for sequencing.

[0131] Nanoparticles

[0132] The invention provides a combination of a closed linear DNA molecule and an mRNA molecule, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0133] The invention also provides a combination comprising: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen; and b. a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen; wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0134] Preferably, the mRNA molecule is comprised in a nanoparticle. Preferably, the closed linear DNA molecule is not comprised in a nanoparticle.

[0135] The nanoparticle may comprise a cargo. The nanoparticle may comprise a cargo and at least one other component of the vaccine composition.

[0136] The cargo may be a closed linear DNA molecule as described herein. The cargo may be an mRNA molecule as described herein.

[0137] The nanoparticle may comprise (a) a lipid component, wherein the lipid component is one or more ionizable lipids and / or one or more cationic lipids; (b) a phospholipid; (c) steroid lipid; and (d) a cationic polymer.

[0138] The nanoparticle may comprise (a) a cargo (i.e. a closed linear DNA molecule as described herein or an mRNA molecule as described herein); (b) a lipid component, wherein the lipid component is one or more ionizable lipids and / or one or more cationic lipids; (c) a phospholipid; (d) steroid lipid; and (e) a cationic polymer.

[0139] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens; wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle, wherein the nanoparticle comprises: (a) a lipid component, wherein the lipid component is one or more ionizable lipids and / or one or more cationic lipids; (b) a phospholipid; (c) steroid lipid; and (d) a cationic polymer.

[0140] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least twenty neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least twenty neoantigens; wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle, wherein the nanoparticle comprises: (a) a lipid component, wherein the lipid component is one or more ionizable lipids and / or one or more cationic lipids; (b) a phospholipid; (c) steroid lipid; and (d) a cationic polymer.

[0141] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least forty neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least forty neoantigens; wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle, wherein the nanoparticle comprises: (a) a lipid component, wherein the lipid component is one or more ionizable lipids and / or one or more cationic lipids; (b) a phospholipid; (c) steroid lipid; and (d) a cationic polymer.

[0142] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen; and b. a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen, wherein the mRNA molecule is comprised in a nanoparticle, wherein the nanoparticle comprises: (a) a lipid component, wherein the lipid component is one or more ionizable lipids and / or one or more cationic lipids; (b) a phospholipid; (c) steroid lipid; and (d) a cationic polymer.

[0143] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens, wherein the mRNA molecule is comprised in a nanoparticle, wherein the nanoparticle comprises: (a) a lipid component, wherein the lipid component is one or more ionizable lipids and / or one or more cationic lipids; (b) a phospholipid; (c) steroid lipid; and (d) a cationic polymer.

[0144] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least twenty neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least twenty neoantigens, wherein the mRNA molecule is comprised in a nanoparticle, wherein the nanoparticle comprises: (a) a lipid component, wherein the lipid component is one or more ionizable lipids and / or one or more cationic lipids; (b) a phospholipid; (c) steroid lipid; and (d) a cationic polymer.

[0145] The combination may comprise: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least forty neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least forty neoantigens, wherein the mRNA molecule is comprised in a nanoparticle, wherein the nanoparticle comprises: (a) a lipid component, wherein the lipid component is one or more ionizable lipids and / or one or more cationic lipids; (b) a phospholipid; (c) steroid lipid; and (d) a cationic polymer.

[0146] The lipid component may comprise an ionizable and / or a cationic lipid. Preferably, the lipid component comprises DLin-MC3-DMA, DLin-KC2-DMA, DLin-DMA, TCL053, SM-102, ALC-0315, C12-200, DODMA, DODAP, Lipid A9, 9A1 P9, Lipid C24, Lipid LP01 , Lipid 5, 4A3-SC8, CKK-E12 (ionizable lipids), DOTMA, DTDTMA, DHDTMA, or DOTAP (cationic lipids). The lipid component may be ALC-0315 and DOTMA. The lipid component may be DLin-MC3-DMA and DOTMA. The lipid component may be ALC-0315 and DLin-MC3-DMA. The lipid component may be SM-102 and DOTAP. The nanoparticle may comprise a combination of one or ionizable lipids. The nanoparticle may comprise one or more cationic lipids. The nanoparticle may comprise one or more ionizable lipids and one or more cationic lipids.

[0147] The phospholipid may comprise DOPE, DOPC, DSPC, DPPC, DMPC, POPC or SOPC. Preferably the phospholipid is DOPE. The phospholipid may be one or more of DOPE, DOPC, DSPC, DPPC, DMPC, POPC or SOPC. The nanoparticle may comprise a steroid lipid. A steroid lipid may comprise cholesterol, or a derivative thereof (a cholesterol derivative), such as beta-sitosterol, fucosterol, campesterol, stigamstanol (alkyl steroids), secosteroids (vitamin D2, D3) or pentacyclic steroids. The steroid lipid may be one or more of cholesterol, beta-sitosterol, fucosterol, campesterol, stigamstanol (alkyl steroids), secosteroids (vitamin D2, D3) or pentacyclic steroids.

[0148] The nanoparticle may comprise a cationic polymer. The cationic polymer may be a polycationic peptide. The polycationic peptide may comprise a nucleic acid-binding cationic component. The polycationic peptide may be a nucleic acid-binding cationic component. The polycationic peptide may comprise a closed linear DNA-binding cationic component. The polycationic peptide may be a closed linear DNA-binding cationic component. The polycationic peptide may comprise an mRNA-binding cationic component. The polycationic peptide may be an mRNA-binding cationic component. The nucleic acid-binding cationic component may be oligolysine.

[0149] The cationic polymer may be a nucleic-acid binding cationic polymer. A cationic component of the cationic polymer may be used to establish a desired charge (i.e. and negative / positive ratio) of the nanoparticle. A specific charge (i.e. a nitrogen / phosphate ratio) may be required to facilitate or enhance cell transfection.

[0150] The nucleic acid-binding cationic polymer may comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 32, at least 34, at least 36, at least 38, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 cationic monomers. Preferably, the nucleic acidbinding cationic polymer comprises at least 16, at least 17 or at least 30 cationic monomers. The nucleic acid-binding cationic polymer may comprise less than 10, or less than 9 cationic monomers. The nucleic acid-binding cationic polymer may comprise 8 cationic monomers.

[0151] The cationic polymer may comprise a lysine, a histidine, or an arginine. The nucleic acid-binding cationic polymer may comprise an oligolysine (linear or branched), an oligohistidine (linear or branched) or an oligoarginine (linear or branched). For example, the nucleic acid-binding cationic polymer may comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 32, at least 34, at least 36, at least 38, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 lysine residues. Preferably, the nucleic acid-binding cationic polymer comprises at least 16, at least 17, or at least 30 lysine residues. More preferably still, the nucleic acidbinding cationic polymer comprises at least 17 lysine residues. The nucleic acid-binding cationic polymer may comprise less than 10, or less than 9 lysine residues. The nucleic acid-binding cationic polymer may comprise 8 lysine residues.

[0152] The cationic polymer may be linear or branched. The nucleic acid-binding cationic polymer may be linear or branched. For example, the nucleic acid-binding cationic polymer may comprise at least 16, at least 17, or at least 30 lysine residues in a linear chain. Alternatively, the nucleic acid-binding cationic polymer may comprise at least 16, at least 17, or at least 30 lysine residues in a branched chain. The nucleic acid may be a nucleic acid-binding cationic polymer. The nucleic acid-binding cationic polymer may be linear or branched. For example, the nucleic acid-binding cationic polymer (e.g. the DNA-binding cationic polymer) may comprise at least 16, at least 17, or at least 30 lysine residues in a linear chain. Alternatively, the nucleic acid-binding cationic polymer (e.g. the DNA- binding cationic polymer) may comprise at least 16, at least 17, or at least 30 lysine residues in a branched chain.

[0153] The cationic polymer may comprise oligolysine (linear or branched) such as K16, K17 or K30, oligohistidine (linear or branched) or oligoarginine (linear or branched) or combination of oligolysine and oligohistidine, oligohistidine and oligoarginine, oligoarginine and oligolysine, or oligolysine, oligohistidine and oligoarginine, or PEI. Preferably, the cationic polymer is oligolysine comprising 16, 17 or 30 lysine residues (K16, K17 or K30). The cationic polymer may consist of K16, K17 or K30.

[0154] The nanoparticle may comprise a targeting moiety.

[0155] The lipid component (one or more ionizable and / or one or more cationic lipids) together with the phospholipid and the steroid lipid (and optionally a PEG lipid) may form a liposome.

[0156] The liposome may comprise at least 1%, at least 2%, at least 3%, at least 4% at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20% at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 30% at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% a cholesterol or derivative thereof (as defined by molar amount of a cholesterol or derivative thereof). That is to say that the liposome may comprise at least 1%, at least 2%, at least 3%, at least 4% at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20% at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% a cholesterol or derivative thereof and at least 99%, at least 98%, at least 97%, at least 96% at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85%, at least 84%, at least 83%, at least 82%, at least 81%, at least 80% at least 79%, at least 78%, at least 77%, at least 76%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, or at least 45% of other lipids in the liposome (as defined by molar ratio). The liposome may comprise 10% cholesterol or derivative thereof. The liposome may comprise 20% cholesterol or derivative thereof. The liposome may comprise 30% cholesterol or derivative thereof. The liposome may comprise 40% cholesterol or derivative thereof.

[0157] The cholesterol derivative may be cholesterol, beta-sitosterol, fucosterol, campesterol, stigamstanol (alkyl steroids), secosteroids (vitamin D2, D3) or pentacyclic steroids. The structures of fucosterol, campesterol and stigamstanol are shown below (from Nano Lett. 2020, 20, 6, 4543-4549):

[0158] The lipid component (one or more ionizable and / or one or more cationic lipids) together with the phospholipid and the steroid lipid (and optionally a PEG lipid) may form a liposome.

[0159] The lipid component may comprise a cationic lipid or an ionizable lipid (or a combination thereof).

[0160] The ionizable lipid may be any one or more of those listed in the table below:

[0161] 31

[0162] 17044091 OMB OMB

[0163] The liposome may comprise one or more of the following cationic lipids: DTDTMA (ditetradecyl trimethyl ammonium), DOTMA (2,3-dioleyloxypropy1-1-trimentyl ammonium), or DHDTMA (dihexadecyl trimethyl ammonium), or DOTAP (1 ,2-dioleoyl-3-trimethylammonium-propane (chloride salt), DOTAP). In addition to the cation, the cationic lipids may comprise a counter anion, for example, an inorganic counter ion, especially a pharmaceutically acceptable anion such as chloride or bromide.

[0164] The nanoparticle may comprise a phospholipid. The term “phospholipid” refers to a lipid comprising a fatty acid chin and a phosphate group. Phospholipids are typically neutral molecules in that they do not have an overall charge, unlike a cationic lipid, which is positively charged. Phospholipids are typically zwitterionic molecules comprising both positive and negative charged components, but no overall charge. For example, the phospholipid may be DOPE (1 ,2-dioleoyl-sn-glycero-3-phosphoetha- nolamine), DOPC (1 ,2-dioleoyl- sn-glycero-3-phosphoethanoltrimethylamine) DSPC (1 ,2-distearoyl- sn-glycero-3-phosphocholine), DPPC (Dipalmitoylphosphatidylcholine), DMPC (1 ,2-dimyristoyl-sn- glycero-3-phosphocholine), or POPC (1-Palmitoyl-2-oleoylphosphatidylcholine), or SOPC (1-stearoyl- 2-oleoyl-sn-glycero-3-phosphocholine).

[0165] The nanoparticles may comprise a PEGylated lipid. The PEG lipid may be provided by the phospholipid comprising a PEG (polyethylene glycol) moiety. The PEG moiety may have a molecular weight of from about 100 to about 10,000, optionally the PEG moiety has a molecular weight of from about 250 to about 7,500, optionally the PEG moiety has a molecular weight of from about 500 to about 5,000, optionally the PEG moiety has a molecular weight of from about 750 to about 4,000, optionally the PEG moiety has a molecular weight of from about 1 ,000 to about 3,000, optionally the PEG moiety has a molecular weight of approximately 2,000.

[0166] The molar ratio of the lipid component comprising a ionizable lipid and / or a cationic lipid to a phospholipid in the nanoparticle may be 1 :1 , 1 :2, 1 :3, 1 :4, 1 :5, 2:1 , 3:1 , 4:1 , 5:1 ,6:1 , 7:1 , 8:1 , 9:1 or

[0167] 32

[0168] 17044091 OMB OMB 10:1. Preferably, the molar ratio of at least one cationic lipid to at least one phospholipid in the nanoparticle is 1 :1 or 2:1 . For example, the molar ratio of DOTMA to DOPE in the nanoparticle may be 1 :1 . That is to say that the molar amount of DOTMA and DOPE in the nanoparticle is the same. The molar ratio of DOTMA to DOPE in the nanoparticle may be 2:1 . That is to say that the molar amount of DOTMA is twice the molar amount of DOPE. The molar ratio of ALC-0315 to DOPE may be 1 :1 , 2:1 , 3:1 , 4:1 , 5:1 ,6:1 , 7:1 , 8:1 , 9:1 or 10:1 . Accordingly, the molar ratio of ALC-0315 to DOTMA to DOPE may be 1 :1 :1 , 2:1 :1 , 3:1 :1 , 4:1 :1 , 5:1 :1 , 6:1 :1 , 7:1 :1 , 8:1 :1 , 9:1 :1 or 10:1 :1. The molar ratio of ALC-0315 to DOTMA to DOPE may be 1 :2:1 . 2:2:1 , 3:2:1 , 4:2:1 , 5:2:1 , 6:2:1 , 7:2:1 , 8:2:1 , 9:2:1 or 10:2:1 . The molar ratio of ALC-0315 to DOTMA to DOPE may be 1 to 10:1 to 2:1 . Preferably, the molar ratio of ALC-0315 to DOTMA is from 1 :1 to 3:1. The ratio of ionisable + cationic (e.g. ALC-0315 + DOTMA to DOPE may be from 3:1 to 10:1 , 4:1 to 8:1 or 5:-6:1 . For example, the ratio of ALC-0315:DOTMA:DOPE may be 3:3:1.

[0169] The mass ratio of cationic polymer to either a closed linear DNA molecule or an mRNA molecule in the nanoparticle may be from 0.1 :1 to 9:1 (cationic polymer : nucleic acid cargo). The mass ratio may be from 1 :1 to 6:1 . The mass ratio may be from 2:1 to 4:1 . For example, the mass ratio of cationic polymer to either a closed linear DNA molecule or an mRNA molecule may be about 0.1 :1 , about 0.5:1 , about 1 :1 , about 1 .5:1 , about 2:1 , about 2.5:1 , about 3:1 , about 3.5:1 , about 4:1 , about 4.5:1 , about 5:1 , about 5.5:1 , about 6:1 , about 6.5:1 , about 7:1 , about 7.5:1 , about 8:1 , about 8.5:1 or about 9:1 [cationic polymer: nucleic acid cargo].

[0170] The molar ratio of cationic polymer to either a closed linear DNA molecule or an mRNA molecule in the nanoparticle may be at least 50:1 , at least 100:1 , at least 150:1 , at least 200:1 , at least 250:1 at least 300:1 , at least 350:1 , at least 400:1 , at least 450:1 . at least 500:1 , at least 550:1 at least 600:1 , at least 650:1 , at least 700:1 , at least 750:1 at least 800:1 , at least 850:1 , at least 900:1 , at least 950:1 at least 1000:1 , at least 1050:1 , at least 1100:1 , at least 1150:1 at least 1200:1 , at least 1250:1 , or at least 1300:1 . The molar ratio may be from 100:1 to 1500:1 , from 200:1 to 1200:1 , from 300:1 to 1100:1 , from 400:1 to 1000:1 , from 500:1 to 900:1 , from 600:1 to 800:1 or about 700:1.

[0171] The molar ratio of the cationic polymer to either a closed linear DNA molecule or an mRNA molecule in the nanoparticle may be at least 350:1 or at least 650:1 and the N / P ratio (charge ratio (i.e. Nitrogen / Phosphate molar ratio) of the nanoparticle may be about 4. The molar ratio of the cationic polymer to the nucleic acid molecule may be from 100:1 to 650:1 or from 200:1 to 400:1 and the N / P ratio of the nanoparticle may be about 4.

[0172] The molar ratio of the cationic polymer to either a closed linear DNA molecule or an mRNA molecule in the nanoparticle may be at least 500:1 or at least 900:1 and the N / P ratio of the nanoparticle may be about 6. The molar ratio of the cationic polymer to the nucleic acid molecule may be from 200:1 to 1100:1 or from 350:1 to 700:1 and the N / P ratio of the nanoparticle may be about 6. The molar ratio of the cationic polymer to either a closed linear DNA molecule or an mRNA molecule in the nanoparticle may be at least 750:1 or at least 1400:1 and the N / P ratio of the nanoparticle may be about 8.

[0173] The molar ratio of the cationic polymer to either a closed linear DNA molecule or an mRNA molecule in the nanoparticle may be from 550:1 to 1400:1 , or from 650:1 to 1100:1 and the N / P ratio of the nanoparticle is 9.

[0174] The cationic polymer may comprise at least 30 (e.g. 31) positively charged amino acids. In such cases, the peptide : DNA or RNA cargo molar ratio may be between 30:1 and 1000:1 , 75:1 to 750:1 or between 100:1 and 500:1.

[0175] The ratios of each of the components of the nanoparticle may be as in any of the nanoparticle formulations set out in WO2024 / 160962, the content of which is incorporated herein by reference in its entirety.

[0176] The nanoparticle (e.g. the non-viral transfection complex) may have a particle size of less than 300 nm, for example less than 200 nm or less than 100 nm or about 80nm.

[0177] The charge ratio (N / P ratio) is calculated from the molar amount of each free amine group(s) (N) in the components of the nanoparticle to the phosphate groups (P) in the components of the nanoparticle. For example, the free amine group(s) may come from the cationic polymer and the lipid component and the phosphate group(s) may come from the phosphate groups in the nucleic acid molecule (e.g. DNA molecule) (P). The charge ratio is typically driven by the mass of the cationic polymer. An N / P ratio of 1 , for example, consists of 1 amine group to 1 phosphate and is conventionally expressed as N / P = 1 . Similarly, a ratio N / P = 5 refers to the ratio between 5 amine groups to 1 phosphate group.

[0178] For example, for nanoparticles consisting of lipid, peptide and mRNA components, the N / P ratio is calculated as followed:

[0179] For three-component nanoparticles, the mass of each component to formulate can be calculated from the desired mass of nucleic acid to be encapsulated and the desired charge ratio of Peptide / mRNA and Lipid / mRNA.

[0180] First, P must be calculated:

[0181] 34

[0182] 17044091 OMB OMB

[0183] The number of moles required of each lipid and peptide in the final formulation can then be calculated as followed:

[0184] Where Npep is equal to the number of positively charged amino acids in the peptide sequence (Lysine, Histidine and Arginine), and NHPis equal to the number of free amine groups in the cationic lipid component.

[0185] Therefore, the mass of peptide or lipid to formulate in the final nanoparticle formulation can be calculated as follows:

[0186] Mass = Moles x Molecular Weight

[0187] Finally, the N / P ratio of the final nanoparticle equation can be calculated using the formulation above.

[0188] The term “about” as used herein for numerical parameters refers to a value within 10% of the underlying parameter (i.e. plus or minus 10%). For example, a charge ratio of “about 4.5” can include charge (N / P) ratios between 4.1 - 5.0, including charge ratios 4.1 and 5.0.

[0189] In a preferred “Hermes 1 ” embodiment of a nanoparticle, the cargo is mRNA and the nanoparticle comprises 50.57% ionizable lipid, 40% Cholesterol, 8.43% phospholipid and 1 % DMG-PEG. The mRNA: lipid mass ratio is 1 :22. The peptide is K16. The total N / P ratio of the mRNA lipid-peptide nanoparticle is 6.08. The ionizable lipid may be ALC-0315, SM-102 or Dlin-MC3-DMA. The phospholipid may be DOPE or DSPC. The PEG-lipid is preferably DMG-PEG.

[0190] In a preferred “Hermes 3” embodiment of a nanoparticle, the cargo is a closed linear DNA molecule and the nanoparticle comprises DOTMA, DOTAP, ALC-0315, SM-102 and / or DLin-MC3-DMA as the cationic or ionizable lipid. The molar ratio of lipids is 48.85% cationic / ionizable lipid, 40% Cholesterol, 8.14% DSPC and 3% DMG-PEG. The mass ratio of a closed linear DNA molecule: lipid is 1 :22. The cationic peptide is K16. The total nitrogen / phosphate ratio (N / P ratio) is 5.8. The nanoparticle may comprise both a cationic and ionizable lipid, preferably at a ratio of 2.35:1 . The total molar ratio of the cationic / ionizable component is preferably 48-51 %.

[0191] 35

[0192] 17044091 OMB OMB Thus, the nanoparticle described herein, preferably, comprises 40% cholesterol. Preferably, the mass ratio of a nucleic acid cargo to lipids in a nanoparticle is about 1 :22. The total nitrogen / phosphate ratio (N / P ratio) may be between 5.5 and 6.2.

[0193] The nanoparticle may comprise:

[0194] The nanoparticle is preferably a self-assembled nanoparticle. The nanoparticle may be a nanoparticle which is produced by a process in which pre-existing components (e.g. a lipid component, a cargo and a cationic polymer) form an organized structure as a consequence of specific, local interactions among the components themselves, without external direction. The nanoparticle may be a nanoparticle that is prepared without pre-prepared liposomes using a microfluidics method, such as a dilution cartridge method in a one step process

[0195] The first vaccine composition, the second vaccine composition, and any subsequent vaccine compositions, if present, may be delivered to a subject by any acceptable route. For example, the vaccine compositions may be delivered by electroporation. Electroporation is the process of using an electric pulse to introduce DNA or RNA into cells by creating temporary pores in the cell membrane. For example, the closed linear DNA molecule as described herein may be delivered to a subject by electroporation. For example, the mRNA molecule as described herein may be delivered to a subject by electroporation. Preferably, the closed linear DNA molecule as described herein is delivered to a subject by electroporation. The vaccine compositions may be delivered by administration of the nanoparticle as described herein. The vaccine compositions which comprise nanoparticles may be delivered by any appropriate route of administration. Vaccine compositions comprising nanoparticles may be administered by topical, oral, rectal, nasal or parenteral (such as intravenous, intradermal, subcutaneous, or intramuscular) routes. Preferably, the vaccine composition comprising the mRNA molecule is delivered to a subject in a nanoparticle.

[0196] Medical applications and administration

[0197] The inventors tested different combinations of the closed linear DNA molecule and the mRNA molecule. The combination comprising a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen and a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen, wherein the mRNA molecule is comprised in a nanoparticle as described herein is denoted as combination “6” in Figure 9. Combination “6” showed a good level of the immune response in mice as well as improved survival of the mice suffering from cancer. It is to be understood that the embodiments in which a first vaccine composition comprises an mRNA molecule encoding at least one neoantigen and a second vaccine composition comprising a closed linear DNA molecule encoding at least neoantigen are also envisaged. In this combination, for example, the RNA molecule may be comprised in a nanoparticle as described herein. This combination is denoted as, for example, “3” in Figure 9. Combination “3” showed a good level of the immune response in mice (see e.g. CD4+ driven TNFa).

[0198] The invention provides a combination or kit-of-parts comprising (a) a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen; and (b) a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen, for simultaneous, separate or sequential use in treating or preventing cancer in a subject.

[0199] The first vaccine composition and the second vaccine composition may be administered or delivered to the subject in any order. Preferably, the first vaccine composition is administered (or delivered) first, before the second vaccine composition.

[0200] The closed linear DNA molecule and / or the mRNA molecule may be comprised in a nanoparticle. Preferably, the mRNA molecule is comprised in a nanoparticle.

[0201] A combination or kit-of-parts comprising (a) a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens; and (b) a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens, may be for simultaneous, separate or sequential use in treating or preventing cancer in a subject, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0202] The combination, kit or kit-of-parts may further comprise an immune checkpoint inhibitor. Thus, the combination or kit-of-parts of (a) a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens; (b) a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens; and (c) an immune checkpoint inhibitor, may be for simultaneous, separate or sequential use in treating or preventing cancer in a subject, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0203] The invention also provides a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen for use in treating or preventing cancer, wherein the first vaccine composition is administered to a subject simultaneously, separately or sequentially with a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle. A first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens may be used in treating or preventing cancer, wherein the first vaccine composition is administered to a subject simultaneously, separately or sequentially with a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0204] The invention also provides a second vaccine composition comprising a mRNA molecule encoding at least one neoantigen for use in treating or preventing cancer, wherein the second vaccine composition is administered to a subject simultaneously, separately or sequentially with a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0205] A second vaccine composition comprising a mRNA molecule encoding at least two neoantigens may be used in treating or preventing cancer, wherein the second vaccine composition is administered to a subject simultaneously, separately or sequentially with a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0206] The invention also provides a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen for use in a heterologous prime-boost immunization regimen in a subject, wherein the first vaccine composition is administered at least once to the subject, and wherein a second vaccine composition, comprising an mRNA molecule encoding at least one neoantigen is separately administered to the subject following at least one administration of the first vaccine, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0207] A first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens may be used in a heterologous prime-boost immunization regimen in a subject, wherein the first vaccine composition is administered at least once to the subject, and wherein a second vaccine composition, comprising an mRNA molecule encoding at least two neoantigens is separately administered to the subject following at least one administration of the first vaccine, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0208] The invention also provides a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen for use in treating or preventing cancer in a subject, wherein the first vaccine composition is administered at least once to the subject, and wherein a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen is separately administered to the subject following at least one administration of the first vaccine, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle. A first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens may be used in treating or preventing cancer in a subject, wherein the first vaccine composition is administered at least once to the subject, and wherein a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens is separately administered to the subject following at least one administration of the first vaccine, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0209] The invention also provides a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen for use in a heterologous prime-boost immunization regimen in a subject, wherein the second vaccine composition is administered to the subject following at least one administration of a first vaccine composition, and wherein the first vaccine composition comprises a closed linear DNA molecule encoding at least one neoantigen, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0210] A second vaccine composition comprising an mRNA molecule encoding at least two neoantigens may be used in a heterologous prime-boost immunization regimen in a subject, wherein the second vaccine composition is administered to the subject following at least one administration of a first vaccine composition, and wherein the first vaccine composition comprises a closed linear DNA molecule encoding at least two neoantigens, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0211] The invention also provides a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen for use in treating or preventing cancer in a subject, wherein the second vaccine composition is administered to the subject following at least one administration of a first vaccine composition, and wherein the first vaccine composition comprises a closed linear DNA molecule encoding at least one neoantigen, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0212] A second vaccine composition comprising an mRNA molecule encoding at least two neoantigens may be used in treating or preventing cancer in a subject, wherein the second vaccine composition is administered to the subject following at least one administration of a first vaccine composition, and wherein the first vaccine composition comprises a closed linear DNA molecule encoding at least two neoantigens, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0213] It is to be understand that, in some embodiments, the second vaccine composition comprising an mRNA molecule encoding at least one (preferably at least two) neoantigens is administered to a subject before the first vaccine composition comprising a closed linear DNA molecule encoding at least one (preferably at least two) neoantigens. Thus, the invention provides a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen for use in a heterologous prime-boost immunization regimen in a subject, wherein the second vaccine composition is administered at least once to the subject, and wherein a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen is separately administered to the subject following at least one administration of the second vaccine, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0214] The invention also provides a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen for use in treating or preventing cancer in a subject, wherein the second vaccine composition is administered at least once to the subject, and wherein a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen is separately administered to the subject following at least one administration of the second vaccine, and wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0215] The description of the embodiments in which the first vaccine composition is administered to the subject first, before the administration of the second vaccine composition apply mutatis mutandis to the embodiments in which the second vaccine composition is administered to the subject first, before the administration of the first vaccine composition. Thus, the skilled person would understand that the first vaccine composition and the second vaccine composition can be administered to the subject in any order. Preferably, the first vaccine composition is administered to the subject before the second vaccine composition is administered to the subject.

[0216] Preferably, the mRNA molecule encodes at least 20 (e.g. at least 40) neoantigens. Preferably, the closed linear DNA molecule encodes at least 20 (e.g. at least 40) neoantigens.

[0217] Preferably, the mRNA molecule is comprised in a nanoparticle as described herein. Preferably, the closed linear DNA molecule is not comprised in a nanoparticle as described herein. The nanoparticle may comprise: (a) a lipid component, wherein the lipid component is one or more ionizable lipids and / or one or more cationic lipids; (b) a phospholipid; (c) steroid lipid; and (d) a cationic polymer.

[0218] The first vaccine composition and a second vaccine composition may be administered to a subject according to a heterologous prime-boost immunization regimen. Thus, in general terms, the invention provides a combination of first and second vaccine compositions as described herein for use in a heterologous prime-boost immunization regimen in a subject. Preferably, the first vaccine composition is administered before the second vaccine composition. The first vaccine composition may be administered at least once, or at least twice to the subject. The second vaccine composition may be administered at least once to the subject. Administration of first and / or second vaccine compositions is intended to stimulate the immune system to produce anti-cancer immune responses. Administration of first and / or second vaccine compositions is intended to stop or reverse tumour growth in a subject. Accordingly, administration of first and / or second vaccine compositions is intended to treat, prevent, induce a protective immune response against or alleviate the symptoms associated with cancer. The vaccine compositions can thus have both prophylactic and therapeutic applications. The subject may be a mammal, preferably a human.

[0219] The cancer may be a lung cancer, a breast cancer (e.g. a triple negative breast cancer), a skin cancer, a colorectal (i.e. colon) cancer, a cervix cancer, a gastric cancer, a urotelial cancer, an ovarian cancer, a glioblastoma cancer, a liver cancer, a uterus cancer, a prostate cancer, a head and neck cancer or leukaemia (e.g. acute lymphoblastic leukaemia).

[0220] Accordingly, the invention provides a method of treating, preventing, inducing a protective immune response against or alleviating the symptoms associated with cancer in a subject, the method comprising administering the combination of a first vaccine composition as described herein and a second vaccine composition as described herein to the subject.

[0221] More specifically the invention provides a vaccination method (comprising a heterologous prime-boost immunization regimen in a subject), the method comprising administering to the subject a first vaccine composition as described herein at least once to the subject and further administering a second vaccine composition as described herein to the subject.

[0222] Similarly, the invention provides a method of treating, preventing, inducing a protective immune response against or alleviating the symptoms associated with cancer in a subject the method comprising administering to the subject a first vaccine composition as described herein at least once to the subject and further administering a second vaccine composition as described herein to the subject.

[0223] The vaccine compositions of the invention may be administered to the subject by any appropriate route of administration. As the skilled person would be aware, vaccine compositions may be administered by topical, oral, rectal, nasal or parenteral (such as intravenous, intradermal, subcutaneous, or intramuscular) routes. In addition, vaccine compositions may be incorporated into sustained release matrices such as biodegradable polymers, the polymers being implanted in the vicinity of, or in close proximity to, where delivery is desired.

[0224] Preferably, the first and second (and any subsequent) vaccine compositions are administered separately. Thus, so-called prime-boost regimens are employed according to the invention. The first vaccine composition is administered at least once to the subject and the second vaccine composition is administered separately at least once to the subject. If present, the third vaccine composition is also administered at least once to the subject.

[0225] The first and / or second (and any subsequent) vaccine compositions may each be administered multiple times to the same subject. The first vaccine composition may be administered at least two, or at least three times to the subject. The first vaccine composition may be administered at least once to the subject before the second vaccine composition is administered to the subject. The second vaccine composition may be administered at least once, at least twice or at least three times to the subject. Thus, the regimen may preferably comprise one administration of the first vaccine composition followed by one, two or three administrations of the second vaccine composition to the subject. This administration pattern may then be repeated if needed and appropriate, for example after monitoring the disease status of the subject or monitoring levels of immune response of the subject over time.

[0226] Administration of each vaccine composition is separated by a suitable period of time, based on the immune response stimulated by the preceding administration of a vaccine composition as would be readily appreciated by the skilled person. Administration of each vaccine is typically separated by an intervening period of at least 1 week and often at least 2 or at least 3 weeks. In some embodiments, administration of each vaccine is separate by a period of around 1-12 months. The periods between administrations may be the same i.e. a regular administration schedule, such as one administration per year. However, different periods with the ranges specified may be applied where appropriate.

[0227] The periods between administration may take account of monitoring of the subject, for example after monitoring the disease status of the subject or monitoring levels of immune response of the subject over time.

[0228] The first vaccine composition may be delivered (or administered) to a subject at a first time point, and the second vaccine composition is delivered (or administered) to the subject at a second time point. The second time point may be at least 14 days after the first time point. If a third vaccine composition is delivered (or administered) to a subject, the third vaccine composition may be delivered (or administered) to a subject at a third time point. The third time point may be at least 14 days after the second time point.

[0229] The relevant dosage of the first and second vaccine compositions can be determined by one skilled in the art and may be adjusted according to factors known in the art such as the symptoms, age and body weight of the subject. Dosage may also be adjusted after the first vaccination on an individual basis, dependent upon variables such as whether or not there are any adverse reactions and based on monitoring of the subject, for example after monitoring the disease status of the subject or monitoring levels of immune response of the subject over time. Heterologous prime-boost immunization requires that a different vaccine is used in the primary and in at least some of the follow up immunizations. As demonstrated herein a closed linear DNA molecule encoding at least one neoantigen and an mRNA molecule encoding at least one neoantigen when administered by homologous prime-boost regimen improved immune response and a reduction in tumor growth in an animal model. Without wishing to be bound by a specific hypothesis, it is likely that the delivery of at least one of the closed linear DNA molecule and the mRNA molecule to the subject in a nanoparticle is responsible for the observed beneficial effects. Specifically, the encapsulation of the mRNA molecule in a nanoparticle and delivery as a boost (after the closed linear DNA molecule was used as a primer) triggers the best response. The vaccine compositions of the invention represent a powerful new therapeutic option for prevention and treatment of cancer.

[0230] In the therapeutic used and methods described herein, the first vaccine composition and the second vaccine composition (and subsequent vaccine compositions) may be administered to a subject in combination with an immune checkpoint inhibitor. The immune checkpoint inhibitor may be administered to a subject before or after the first vaccine composition is administered. The immune checkpoint inhibitor may be administered to a subject before and after the first vaccine composition is administered. The immune checkpoint inhibitor may be administered to a subject before or after the second vaccine composition is administered. The immune checkpoint inhibitor may be administered to a subject before and after the second vaccine composition is administered. Preferably, the immune checkpoint inhibitor is administered to the subject before the first vaccine and second vaccine compositions are administered. The immune checkpoint inhibitor may be administered to a subject on a regular basis throughout the immunization with the combination as described herein. The immune checkpoint inhibitor may be an anti-CTLA-4 molecule, an anti-PD-1 molecule, an anti-PD-L1 molecule, an anti-LAG-3 molecule. Preferably, the immune checkpoint inhibitor is an anti-CTLA-4 molecule.

[0231] Kits

[0232] The invention further provides a kit comprising a first and second vaccine compositions as described herein. The invention provides a kit-of-parts comprising (a) a first vaccine composition comprising a closed linear DNA molecule encoding at least one neoantigen; and (b) a second vaccine composition comprising an mRNA molecule encoding at least one neoantigen.

[0233] The kit-of-parts may comprise (a) a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens; and (b) a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens.

[0234] The kit or kit-of-parts may comprise (a) a first vaccine composition comprising a closed linear DNA molecule encoding at least twenty neoantigens; and (b) a second vaccine composition comprising an mRNA molecule encoding at least twenty neoantigens. The kit or kit-of-parts may further comprise a third (or subsequent) vaccine composition. The kit or kit- of-parts may further comprise an immune checkpoint inhibitor.

[0235] The closed linear DNA molecule and / or the mRNA molecule may be comprised in a nanoparticle. Preferably, the mRNA molecule is comprised in a nanoparticle.

[0236] The first and second vaccine compositions are provided may be provided in separate containers. Separate kits, for example with separate product labels or instructions for use, may be provided with each type of vaccine composition (i.e. first and second vaccine compositions as described herein).

[0237] The kits of the invention are for use in a heterologous prime-boost immunization regimen in a subject. The kits of the invention are for use in treating, preventing, inducing a protective immune response against or alleviating the symptoms associated with cancer in a subject.

[0238] All such kits may be provided with suitable instructions for use. The instructions for use may explain the administration schedule for the compositions. The kits may therefore comprise instructions for administering the first and second vaccine compositions as described herein. The kits may comprise multiple unit doses of the vaccine compositions. The instructions for use may further explain the storage conditions for the compositions, particularly during the time period between administration of the doses of the vaccine compositions. These kits may be applied to all relevant methods of the invention as disclosed herein.

[0239] The invention is further defined in the following clauses:

[0240] 1 . A combination comprising: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens; wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0241] 2. The combination of clause 1 , wherein the mRNA molecule is comprised in a nanoparticle, and optionally the closed linear DNA molecule is delivered to a subject by electroporation.

[0242] 3. The combination of clause 1 or clause 2, wherein the closed linear DNA molecule encodes at least three, at least four, at least five, at least 10, at least 20, at least 30, or at least 40 neoantigens. The combination of any one of clauses 1-3, wherein the mRNA molecule encodes at least three, at least four, at least five, at least 10, at least 20, at least 30, or at least 40 neoantigens. The combination of any one of clauses 1-4, wherein the closed linear DNA and the mRNA molecule encode at least 1 , at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, or at least 40 of the same neoantigens. The combination of any one of clauses 1-5, wherein the nanoparticle comprises: a. a lipid component, wherein the lipid component is one or more ionizable lipids and / or one or more cationic lipids; b. a phospholipid; c. a steroid lipid; and d. a cationic polymer. The combination of clause 6, wherein the cationic polymer is a polycationic peptide. The combination of clause 7, wherein the polycationic peptide comprises a nucleic acidbinding cationic component, optionally wherein the nucleic acid-binding cationic component is oligolysine. The combination of any one of clauses 1-8, wherein the first vaccine composition is delivered to a subject at a first time point, and the second vaccine composition is delivered to the subject at a second time point. The combination of clause 9, wherein the second time point is at least 14 days after the first time point. The combination of any one of clauses 1-10, wherein the first vaccine composition is delivered to a subject at a first time point by electroporation, and the second vaccine composition, which is comprised in a nanoparticle, is delivered to the subject at a second time point. The combination of any one of clauses 1-11 , wherein the combination comprises a third vaccine composition, wherein the third vaccine composition comprises an mRNA molecule encoding at least two neoantigens. The combination of clause 12, wherein the third vaccine composition is delivered to the subject at a third time point, optionally wherein the third time point is at least 14 days after the second time point. The combination of any one of clauses 1-13, wherein the combination further comprises an immune checkpoint inhibitor. A combination or kit-of-parts comprising (a) a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens; and (b) a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens, for simultaneous, separate or sequential use in treating or preventing cancer in a subject, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle. A first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens for use in treating or preventing cancer, wherein the first vaccine composition is administered to a subject simultaneously, separately or sequentially with a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle. A second vaccine composition comprising a mRNA molecule encoding at least two neoantigens for use in treating or preventing cancer, wherein the second vaccine composition is administered to a subject simultaneously, separately or sequentially with a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle. A first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens for use in a heterologous prime-boost immunization regimen in a subject, wherein the first vaccine composition is administered at least once to the subject, and wherein a second vaccine composition, comprising an mRNA molecule encoding at least two neoantigens is separately administered to the subject following at least one administration of the first vaccine, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle. A first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens for use in treating or preventing cancer in a subject, wherein the first vaccine composition is administered at least once to the subject, and wherein a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens is separately administered to the subject following at least one administration of the first vaccine, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle. 20. A second vaccine composition comprising an mRNA molecule encoding at least two neoantigens for use in a heterologous prime-boost immunization regimen in a subject, wherein the second vaccine composition is administered to the subject following at least one administration of a first vaccine composition, and wherein the first vaccine composition comprises a closed linear DNA molecule encoding at least two neoantigens, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0243] 21 . A second vaccine composition comprising an mRNA molecule encoding at least two neoantigens for use in treating or preventing cancer in a subject, wherein the second vaccine composition is administered to the subject following at least one administration of a first vaccine composition, and wherein the first vaccine composition comprises a closed linear DNA molecule encoding at least two neoantigens, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

[0244] 22. A closed linear DNA molecule, wherein the closed linear DNA molecule encodes at least 40 neoantigens.

[0245] 23. An mRNA molecule, wherein the mRNA molecule encodes at least 40 neoantigens.

[0246] 24. A composition comprising a closed linear DNA molecule, wherein the closed linear DNA molecule encodes at least 40 neoantigen, and wherein the closed linear DNA molecule is encapsulated in a nanoparticle.

[0247] 25. A composition comprising an mRNA molecule, wherein the mRNA molecule encodes at least 40 neoantigen, and wherein the mRNA molecule is encapsulated in a nanoparticle.

[0248] 26. The closed linear DNA molecule of clause 22, the mRNA molecule of clause 23, or the composition of clause 24 or 25, wherein each of the at least 40 neoantigens is different, optionally wherein the at least 40 different neoantigens are breast cancer neoantigens or colorectal cancer neoantigens.

[0249] 27. The composition of any one of clauses 24-26, wherein the nanoparticle comprises: a. a lipid component, wherein the lipid component is one or more ionizable lipids and / or one or more cationic lipids; b. a phospholipid; c. a steroid lipid; and d. a cationic polymer.

[0250] BRIEF DESCRIPTION OF THE FIGURES Figure 1 illustrates the identification of immunogenic neoantigens induced by M20-hpDNA-EP (i.e. a closed linear DNA molecule delivered by electroporation) vaccination. A) Experimental design: C57BI / 6 female mice were immunized by electroporation (EP) on days 0, 14 and 28 with 6.7 pg of a M20-hpDNA-EP construct encoding for 20 neoantigens from MC38 model. 1-week after the last vaccination mice were sacrificed and the spleen was explanted for IFNy ELISpot assay. B) Spot forming colonies (SFC) secreting IFNy upon stimulation with 137 single 15-mer peptide overlapping for 11 residues that code the sequence of M20 vaccine. C) Intra cellular staining (ICS) analysis by flow cytometry of neoantigens identified in the panel B. CD4+and CD8+ T cells were identified by ICS analysis. ICS stands for Intra Cellular Staining.

[0251] Figure 2 illustrates that the induction of neoantigen-specific T cell responses correlates with a significant tumour protection. A) Experimental design. C57BI / 6 female received three immunizations with M20-hpDNA-EP (i.e. a closed linear DNA molecule delivered by electroporation) at the dose of 6,7 mg in a prophylactic setup. One month after the last immunization, mice were inoculated with 3 x 105MC38 tumor cells s.c. in the right flank (n=5) and the tumor size was monitored. Unvaccinated naive mice (n=5) were also challenged with tumour cell line. B) Percentages of IFNy+and TNFa+CD4+and CD8+T cells measured in Peripheral Blood Mononuclear Cells (PBMCs) 1 week after the last vaccination are shown. PBMCs were restimulated with the peptide pool identified in the ELISpot assay. C) Tumor volumes and survival percentage determined over time are shown. The unpaired t- test was used to make comparisons: *P<0.05; **P< 0.01 ; ***P<0.001 vs untreated group. ICS stands for Intra Cellular Staining.

[0252] Figure 3 illustrates the identification of the maximal effective M20-hpDNA-EP (i.e. a closed linear DNA molecule delivered by electroporation) dose. A) Experimental design. C57BI / 6 female received three M20-hpDNA-EP immunizations at escalating doses. One month after the last immunization, mice were inoculated with 3 x 105MC38 tumor cells s.c. in the right flank (n=5) and the tumor size was monitored. Unvaccinated naive mice (n=5) were also challenged with tumour cell line. B) Percentages of IFNy+ / TNFa+ / IL-2+ CD4+ and CD8+ T cells measured in PBMCs 1 week after the last vaccination are shown. PBMCs were restimulated with the peptide pool identified in the ELISpot assay. C) Tumor volumes and survival percentage determined over time are shown. The unpaired t-test was used to make comparisons: *P<0.05; **P< 0.01 vs untreated group. ICS stands for Intra Cellular Staining.

[0253] Figure 4 shows that M20-hpDNA-EP (i.e. a closed linear DNA molecule delivered by electroporation) synergizes with aCTLA-4 in a therapeutic MC38 tumour model. A) Experimental design: C57BI / 6 female mice were inoculated with 3 x 10sMC38 tumor cells s.c. in the right flank. Treatment with aCTLA-4 and vaccine starting from day 2 and 3, respectively. B) Graphs represent tumor volumes determined over time and survival curves. C) Percentages of IFNy+ / TNFcr7IL-2+CD4+and CD8+T cells measured in PBMCs 1 week after the last vaccination are shown. PBMCs were restimulated with

[0254] 48

[0255] 17044091 OMB OMB the peptide pool identified in the ELISpot assay. The unpaired t-test was used to make comparisons: *P< 0.05 vs aCTLA-4 treated mice. ICS stands for Intra Cellular Staining.

[0256] Figure 5 illustrates the identification of immunogenic neoantigens induced by C20 vaccine targeting 20 neoantigens specific for the CT26 tumour model. A) Experimental design: Balb / C female mice were immunized on days 0, 21 and 42 with 6.7 pg of C20-hpDNA-EP (i.e. a closed linear DNA molecule delivered by electroporation). After 1-week mice were sacrificed and the spleen was explanted for immune responses analysis. B) Spot forming colonies (SFC) secreting IFNy measured by ELISPOT assay on splenocytes stimulated by single 15-mer peptides scanning the coding region of the C20 vaccine are shown.

[0257] Figure 6 shows that the C20-hpDNA-EP (i.e. a closed linear DNA molecule delivered by electroporation) induced immune responses correlate with a significant tumour protection. A) Experimental design. BALB / c female mice received three immunizations with EP in a prophylactic setup. One month after the last immunization, mice were inoculated with 1 x 106CT26 tumour cells s.c. in the right flank (n=10) and the tumour size was monitored. Unvaccinated naive mice (n=5) were also challenged with tumour cell line. B) Percentages of IFNy+ / TNFa+CD4+and CD8+T cells measured in PBMCs 1 week after the last vaccination are shown. PBMCs were restimulated with the peptide pool identified in the ELISpot assay. C) Tumour volumes determined over time are shown. D) Mice which remained tumour free were subjected to a second tumour challenge. Graphs show the long-time protective effect of C20 vaccines in a prophylactic vaccination protocol. The unpaired t-test was used to make comparisons: **P< 0.01 ; ***P<0.001 vs untreated group. ICS stands for Intra Cellular Staining.

[0258] Figure 7 shows that the M20 vaccine formulated as an mRNA molecule in a lipid nanoparticle (LNP) (M20-RNA-Hermes1) induces stronger neoantigen specific CD8+T cell responses compared to M20- hpDNA-EP (i.e. a closed linear DNA molecule delivered by electroporation). A) Experimental design. C57BI / 6 female received three immunizations with the M20 vaccines in a prophylactic setup. One month after the last immunization, mice were inoculated with 3 x 105MC38 tumor cells s.c. in the right flank (n=5) and the tumor size was monitored. Unvaccinated naive mice (n=5) were also challenged with tumour cell line. B) Percentages of IFNy+and TNFa+CD4+and CD8+T cells measured in PBMCs 1 week after the second and last vaccination are shown. PBMCs were restimulated with the peptide pool identified in the ELISpot assay. C) Tumor volumes and survival percentage determined over time are shown. D) & E) Mice immunized with M20-RNA-Hermes1 which remained tumour free were subjected to a second tumour challenge. Graph shows the protective effect of M20-RNA-Hermes1 vaccine. The unpaired t-test was used to make comparisons between two vaccine formulations (B) or untreated group (C, D): **P< 0.01 ; ***P<0.001 . ICS stands for Intra Cellular Staining.

[0259] Figure 8 shows that the heterologous M20 vaccinations induced strong and durable CD8+ T cells as lipid nanoparticle (LNP) formulations. A) Experimental design. C57BI / 6 female received three

[0260] 49

[0261] 17044091 OMB OMB immunizations with the different M20 vaccines and prime / boost / boost protocols. B) C) Percentages of IFNy+and TNFa+CD8+T cells measured in PBMCs. PBMCs were restimulated with the peptide pool identified in the ELISpot assay. D) Percentage of naive, active, effector and central CD4+and CD8+T cells memory was measured in PBMCs 1 week after each vaccination. PBMCs were restimulated with the peptide pool identified in the ELISpot assay. Graphs show the percentages of neoantigens- specific CD8+T active and effector memory cells, measured after the last vaccination. E) Representative plots of the different treatments acquired by the Northern Lights flow cytometer. The unpaired t-test was used to make comparisons between the vaccine formulations: *P<0.05; **P< 0.01. In Figure 8, “H1-RNA”, and “H3-RNA” denote an mRNA encapsulated in a nanoparticle, “DNA-EP” denotes a closed linear DNA molecule delivered by electroporation, “RNA-EP” denotes an mRNA molecule delivered by electroporation, “H3-DNA” denotes a closed linear DNA molecule encapsulated in a nanoparticle and “LNP-RNA” denotes an mRNA molecule encapsulated in a formulation known in prior art (see Schoenmaker et al. (2021) International Journal of Pharmaceutics, Vol. 61 , 120586 “mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability). All formulations refer to M20 vaccine. ICS stands for Intra Cellular Staining.

[0262] Figure 9 shows that the M20 heterologous prime / boost vaccination resulted in a stronger tumour protection. A) Experimental design. C57BI / 6 female received three immunizations with the M20 formulations in a prophylactic setup. Twelve days after the last immunization, mice were inoculated with 3 x 105MC38 tumor cells s.c. in the right flank (n=5) and the tumor size was monitored. Unvaccinated naive mice (n=5) were also challenged with tumour cell line. B) Percentages of IFNy+ / TNFa+CD4+and CD8+T cells measured in PBMCs 1 week after last vaccination are shown. PBMCs were restimulated with the peptide pool identified in the ELISpot assay. C) Tumor volumes determined over time is shown. D) The graph represents the percentage of tumor-bearing (black) and tumor-free (gray) mice in each experimental group. The unpaired t-test was used to make comparisons between groups: **P< 0.01 , ***P<0.001 . ICS stands for Intra Cellular Staining.

[0263] Figure 10 illustrates the identification of immunogenic neoantigens induced by E40 (i.e. a closed linear DNA molecule delivered by electroporation or mRNA encapsulated in LNP-Hermes) vaccination. A) Experimental design: C57BI / 6 female mice were immunized by electroporation (EP) on days 0, 21 and 42 with 10 pg of a closed linear DNA molecule construct encoding for the 40 neoantigens from EO771 model. 1-week after the last vaccination mice were sacrificed and the spleen was explanted for IFNy ELISpot assay. B) Spot forming colonies (SFC) secreting IFNy upon stimulation with single 20-mer peptide coding sequence of E40 vaccine. Graph shows peptides resulting positive. C) Experimental design: C57BI / 6 female mice were vaccinated on days 0 and 14 with 10 pg of mRNA molecule construct encoding for the 40 neoantigens from EO771 model. 1-week after the last vaccination mice were sacrificed and the spleen was explanted for IFNy ELISpot assay. D) Spot forming colonies (SFC) secreting IFNy upon stimulation with single 20-mer peptide coding sequence of E40 vaccine. Graph shows peptides resulting positive. E-F) illustrates ICS analysis by flow cytometry of neoantigens identified in the Figure 10. CD4+e CD8+T cells were identified by IFNy

[0264] 50

[0265] 17044091 OMB OMB ICS analysis. The unpaired t-test was used to make comparisons between groups: *P<0.05, **P< 0.01 , ****P<0.0001 . Table 7 summarizes the peptide code, the sequence and the related gene.

[0266] Figure 11 shows that E40 vaccination (i.e. a closed linear DNA molecule delivered by electroporation or a mRNA molecule encapsulated in LNP) induce a significant tumour protection. A) Experimental design. C57BI / 6 female mice received three immunizations with electroporation (EP) in a prophylactic setup. One month after the last immunization, mice were inoculated with 0.2 x 106EO771 tumour cells in mammary fat pad injection (n=10) and the tumour size was monitored. Unvaccinated naive mice (n=5) were also challenged with tumour cell line. B) Tumour volumes and survival curves determined over time are shown. C) Experimental design. C57BI / 6 female mice received two immunizations with E40-mRNA-Hermes in a prophylactic setup. 13 days after the last immunization, mice were inoculated with 0.2 x 106EO771 tumour cells in mammary fat pad injection (n= 10) and the tumour size was monitored. Unvaccinated naive mice (n=5) were also challenged with tumour cell line. D) Tumour volumes and survival curves determined over time are shown. The unpaired t-test was used to make comparisons: *P<0.05, **P< 0.01.

[0267] Figure 12 illustrates the long-time protective effect of E40-hpDNA-EP vaccine in a prophylactic vaccination protocol. A) Experimental design. C57BI / 6 female mice received three immunizations with electroporation (EP) in a prophylactic setup. Two months after the first challenge, mice which remained tumour free after first challenge were subjected to a second tumour challenge (0.2 x 106 EO771 tumour cells in mammary fat pad injection (on the left) (n=10)). Unvaccinated naive mice (n=5) were also challenged with tumour cell line. B) Tumour volumes and survival curves determined over time are shown. The unpaired t-test was used to make comparisons: **P< 0.01 ; ***P<0.001 .

[0268] Figure 13 shows biophysical characteristics of lipid nanoparticle formulations. A) Size, B) polydispersity index (PDI) and C) encapsulation efficiency (EE%) of lipid nanoparticles (termed “Hermes” or “H1 ” or “H3”) or control LNP of prior art encapsulating mRNA or closed linear DNA encoding 20 neoantigens against the MC38 tumour model (M20) or 40 neoantigens against the EO771 tumour model (E40). n = 3.

[0269] The invention will be further understood with reference to the following non-limiting examples:

[0270] EXAMPLES

[0271] Example 1

[0272] 1. Introduction

[0273] A heterologous prime / boost protocol was developed based on synthetic DNA and a new formulation of RNA vaccine. This involved using a synthetic cargo (e.g. a closed linear DNA molecule and / or an mRNA molecule), that was named “Neo-Lin”, to deliver a neoantigen cancer vaccine (NCV).

[0274] 51

[0275] 17044091 OMB OMB A synthetic DNA vaccine candidate encoding twenty neoantigens, was evaluated in both prophylactic and therapeutic vaccination settings. The results demonstrate that Neo-Lin provides comparable protection to plasmid DNA (pDNA) delivered by electroporation (pDNA-EP) in prophylactic vaccination but exhibits reduced efficacy in therapeutic settings. To further improve the therapeutic effect, a heterologous prime-boost platform for delivery of neoantigens has been developed. Specifically, a heterologous prime-boost platform with either a closed linear DNA molecule or as an mRNA molecule formulated in a nanoparticle has been tested. In the Examples, the constructs of the closed linear DNA molecule formulated in the nanoparticle are denoted “H3-DNA” and the constructs of the mRNA molecule formulated in the nanoparticle are denoted “H1-RNA” and “H3-RNA”.

[0276] 2. Methods & Results

[0277] A. Testing of the vaccines comprising either a closed linear DNA molecule and / or an mRNA molecule and delivered by electroporation (i.e. without encapsulation in a nanoparticle)

[0278] To test the capability of Neo-Lin in the preclinical cancer models, two vaccines were generated: M20 for MC38 cancer cells (Yadav, M. et al. Predicting immunogenic tumour mutations by combining mass spectrometry and exome seguencing. Nature 515, 572-6 (2014).) and C20 for CT26 cancer cells (Kreiter, S. et al. Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature 520, 692-696 (2015)). Both cancer cell lines are murine models for colorectal carcinoma. Both tumor models have been extensively utilized in the NCV field (Lang, F., Schrors, B., Lower, M., Tureci, O. & Sahin, U. Identification of neoantigens for individualized therapeutic cancer vaccines. Nat. Rev. Drug Discov. 21 , 261-282 (2022).). For both tumor models a vaccine comprising either a closed linear DNA molecule or an mRNA molecule, each encoding for 20 neoantigens was produced (termed Neo-Lin). Neoantigens were synthesized as peptides between 25 and 28 amino acids in length, positioning the mutated amino acid at the 14th position within the seguence whenever possible to ensure the mutation is recognized by MHC class I or class II, as appropriate.

[0279] For the M20 vaccine design, we drew upon the RNA-seg experiment detailed in the Yadav et al. study (Yadav, M. et al. Predicting immunogenic tumour mutations by combining mass spectrometry and exome seguencing. Nature 515, 572-6 (2014)). Neoantigens were prioritized according to predicted immunogenicity and expression levels, with the top 20 selected (Table 1). Drawing from the rich literature on M38 neoantigens (Yadav, M. et al. Predicting immunogenic tumour mutations by combining mass spectrometry and exome seguencing. Nature 515, 572-6 (2014), Aurisicchio, L. et al. Poly-specific neoantigen-targeted cancer vaccines delay patient derived tumor growth. 4, 1-13 (2019), Brennick, C. A. et al. An unbiased approach to defining bona fide cancer neoepitopes that elicit immune-mediated cancer rejection. J. Clin. Invest. 131 , (2021), Chen, B., Khodadoust, M. S., Liu, C. L., Newman, A. M. & Alizadeh, A. A. Profiling tumor infiltrating immune cells with CIBERSORT. Methods Mol. Biol. 1711 , 243-259 (2018), Liu, L. et al. Concurrent delivery of immune checkpoint blockade modulates T cell dynamics to enhance neoantigen vaccine-generated antitumor immunity. Nat. Cancer 3, 437-452 (2022), D’Alise, A. M. et al. Adenoviral-based vaccine promotes neoantigenspecific CD8+ T cell sternness and tumor rejection. Sci. Transl. Med. 14, (2022)), the M20 vaccine incorporates nine neoantigens (Adpgk, Dpagtl , Repsi , Tmem135, Spire"! , Wbp7, Hacel , Nle1 , Zbtb40) known for their efficacy across multiple independent studies (Yadav, M. et al. Predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing. Nature 515, 572-6 (2014), Aurisicchio, L. et al. Poly-specific neoantigen-targeted cancer vaccines delay patient derived tumor growth. 4, 1-13 (2019), D’Alise, A. M. et al. Adenoviral-based vaccine promotes neoantigen-specific CD8+ T cell sternness and tumor rejection. Sci. Transl. Med. 14, (2022), and Salvatori, E. et al. Neoantigen cancer vaccine augments anti-CTLA-4 efficacy, npj Vaccines 7, 1-10 (2022)). The remaining 11 neoantigens (Hnrnpl, Pop1 , Pam, Actrl b, Yipf4, Huwel , Slc35e4, Gtf2i, Latsl , Ndfip2, Atg9a) are reported also in a more recent publication (Capietto, A. H. et al. Mutation position is an important determinant for predicting cancer neoantigens. J. Exp. Med. 217, (2020)). In constructing the final vaccine, the positioning of each neoantigen was chosen to prevent the formation of immunogenic junctions. Since CD8+T cell responses have been implicated in protection against tumour, a study was first designed to identify MHC class I and class Il-restricted neoantigens.

[0280] Table 1. List of 20 neoantigens in M20 constructs

[0281] The specificity of the immunological response was measured by ex vivo enzyme-linked immunospot (ELISpot) assay conducted using single overlapping ~15-mer peptides spanning full length M20 as

[0282] 53

[0283] 17044091 OMB OMB the stimulant. IFNy ELISpot analysis on splenocytes gave the results depicted in Figure 1 and Table 2.

[0284] Table 2. List of ~15-mer peptides included in M20-pool used for in vitro lymphocyte stimulation.

[0285] 10 out of 137 15-mer peptides induced a positive response and correspond to 6 of the 20 neoantigens identified by the pipeline (showing approximately 25% success in predicting useful neoantigens). Figure 1 illustrates the identification of immunogenic neoantigens induced by M20-hpDNA-EP (i.e. a closed linear DNA molecule delivered by electroporation) vaccination. A) Experimental design: C57BI / 6 female mice were immunized by electroporation (EP) on days 0, 14 and 28 with 6.7 pg of a closed linear DNA molecule encoding for 20 neoantigens from MC38 model. 1-week after the last vaccination mice were sacrificed and the spleen was explanted for IFNy Elispot assay. B) Spot forming colonies (SFC) secreting IFNy upon stimulation with 137 single 15-mer peptide overlapping for 11 residues the coding sequence of M20 vaccine. C) Intra cellular staining (ICS) analysis by flow cytometry of neoantigens identified in the panel B. CD4+and CD8+T cells were identified by ICS analysis.

[0286] A dose response finding was carried out in parallel with the plasmid DNA (pDNA) version of the M20 vaccine (Figure 2). One week after the last vaccination Intra Cellular Staining (ICS) with PBMC revealed a clear dose response as means of both neoantigen-specific IFNy+and TNFa+CD8+T cells. Figure 2 illustrates that the induction of neoantigen-specific T cell response correlates with a significant tumour protection. A) Experimental design. C57BI / 6 female received three immunizations with M20-hpDNA-EP at the dose of 6,7 mg in a prophylactic setup. One month after the last immunization, mice were inoculated with 3 x 105MC38 tumor cells s.c. in the right flank (n=5) and the tumor size was monitored. Unvaccinated naive mice (n=5) were also challenged with tumour cell line. B) Percentages of IFNy+and TNFa+CD4+and CD8+T cells measured in Peripheral Blood Mononuclear Cells (PBMCs) 1 week after the last vaccination are shown. PBMCs were restimulated with the peptide pool identified in the ELISpot assay. C) Tumor volumes and survival percentage determined over time are shown. The unpaired t-test was used to make comparisons: *P<0.05; **P< 0.01 ; ***P<0.001 vs untreated group.

[0287] 54

[0288] 17044091 OMB OMB Tumor challenge on day 72 showed a correlation with the dose of the vaccine and the immune responses. 80% of mice were protected at 6.7 pg of the closed linear DNA molecule 60% at 3.35 pg and 40% at the lowest dose of 1 .68 pg (Figure 2). Using a higher dose of 6.7 pg did not improve antitumor activity (Figure 3). Figure 3 illustrates the identification of the maximal effective M20-hpDNA-EP dose. A) Experimental design. C57BI / 6 female received three M20-hpDNA-EP at escalating doses. One month after the last immunization, mice were inoculated with 3 x 105MC38 tumor cells s.c. in the right flank (n=5) and the tumor size was monitored. Unvaccinated naive mice (n=5) were also challenged with tumour cell line. B) Percentages of IFNy+ / TNFa7IL-2+CD4+and CD8+T cells measured in PBMCs 1 week after the last vaccination are shown. PBMCs were restimulated with the peptide pool identified in the ELISpot assay. C) Tumor volumes and survival percentage determined over time are shown. The unpaired t-test was used to make comparisons: *P<0.05; **P< 0.01 vs untreated group.

[0289] Therapeutic vaccination with the M20-hpDNA in combination with anti-CTLA-4 molecule (aCTLA-4) was conducted starting with the immune checkpoint inhibitor treatment on day 2 and vaccination on day 3 (Figure 4). The combined treatment resulted in a significant antitumor activity (Figure 4B), which was associated with a significant increase of IFN-y+and TNF-a+CD8+T cells. Figure 4 shows that M20-hpDNA-EP synergizes with aCTLA-4 in a therapeutic MC38 tumour model. A) Experimental design: C57BI / 6 female mice were inoculated with 3 x 10sMC38 tumor cells s.c. in the right flank. Treatment with aCTLA-4 and vaccine starting from two day 2 and 3, respectively. B) Graphs represent tumor volumes determined over time and survival curves. C) Percentages of IFNy7TNFc(7IL-2+CD4+and CD8+T cells measured in PBMCs 1 week after the last vaccination are shown. PBMCs were restimulated with the peptide pool identified in the ELISpot assay. The unpaired t-test was used to make comparisons: *P< 0.05 vs aCTLA-4 treated mice.

[0290] Following the same approach, the C20 vaccine was developed on the basis of published information (Table 3).

[0291] 55

[0292] 17044091 OMB OMB

[0293]

[0294] Table 3. List of neoantigens in C20 constructs.

[0295] Analysis of neoantigen-specific immune response by IFNy ELISpot test resulted in the identification of 12 positive 15-mer peptides out of 137, confirming 6 out of 20 predicted neoantigens (showing approximately 30% success in predicting useful neoantigens) (Figure 5B; Table 4). Interestingly, splenocytes secreting IFNy after 15-mer peptides stimulation were CD8+T cells, as expected from the pipeline prediction (data not shown). Figure 5 illustrates the identification of immunogenic neoantigens induced by C20 vaccine targeting 20 neoantigens specific for the CT26 tumour model. A) Experimental design: Balb / C female mice were immunized on days 0, 21 and 42 with 6.7 pg of C20- hpDNA-EP. After 1-week mice were sacrificed and the spleen was explanted for immune responses analysis. B) SFC producing IFNy were measured by ELISpot assay on splenocytes stimulated by single 15-mer peptides scanning the coding region of the C20 vaccine.

[0296] Table 4. List of 15-mer peptides included in C20-pool used for in vitro lymphocyte stimulation.

[0297] To test the impact of C20 vaccination on tumor growth mice were vaccinated 3 times every 3 weeks with C20-hpDNA-EP (i.e. the closed linear DNA molecule delivered by electroporation) and C20- pDNA-EP (i.e. the plasmid DNA delivered by electroporation) as benchmark (Figure 6A). Similar immune responses were observed by flow cytometry in PMBCs 1 week after the last vaccination (Figure 6B). Tumor challenge (1 ° challenge) conducted at 1 month after vaccination showed a significant tumor protection in 60% of C20-hpDNA-EP treated mice (Figure 6D). To confirm that a memory T cell response was induced a second tumor challenge (2° challenge) on the opposite flank was conducted on day 160 resulting in complete tumor protection. Figure 6 shows that the C20- hpDNA-EP induced immune responses correlate with a significant tumour protection. A) Experimental design. BALB / c female mice received three immunizations with EP in a prophylactic setup. One month after the last immunization, mice were inoculated with 1 x 106CT26 tumour cells s.c. in the right flank (n=10) and the tumour size was monitored. Unvaccinated naive mice (n=5) were also challenged with tumour cell line. B) Percentages of IFNy+ / TNFa+CD4+and CD8+T cells measured in PBMCs 1 week after the last vaccination are shown. PBMCs were restimulated with the peptide pool identified in the ELISpot assay. C) Tumour volumes determined over time are shown. D) Mice which remained tumour free were subjected to a second tumour challenge. Graphs show the longtime protective effect of C20 vaccines in a prophylactic vaccination protocol. The unpaired t-test was used to make comparisons: **P< 0.01 ; ***P<0.001 vs untreated group.

[0298] B. Testing of the heterologous prime-boost system, in which one of the vaccines comprised a closed linear DNA molecule and the other of the vaccines an mRNA molecule

[0299] In the first instance, shown in Figure 7, an mRNA molecule was encapsulated in a nanoparticle as described herein and tested against a closed linear DNA molecule delivered by electroporation (without encapsulation in a nanoparticle) with respect to the immune responses and tumor volume.

[0300] In a first experiment M20-hpDNA-EP (i.e. the closed linear DNA molecule delivered by electroporation) was compared to M20-RNA-Hermes1 (i.e. the mRNA molecule comprised in a nanoparticle) in a biweekly vaccination protocol repeated 3 times (Figure 7A).

[0301] The nanoparticles (referred to “Hermes 1” and “Hermes 3” in the Examples) comprise one or more ionizable lipids and / or one or more cationic lipids, a phospholipid, a steroid lipid, a PEG-lipid, and a cationic polymer. Hermes 1 nanoparticle encapsulated mRNA as the cargo. Hermes 3 nanoparticle encapsulated closed linear DNA as the cargo. Formulations were prepared by microfluidic mixing of lipids in ethanol and payload in NaAC (pH 4) at a flow rate ratio of 3:1 and a total flow rate of 12 mL / min. Cationic peptide in water was placed in an in-line dilution channel and mixed with lipid / payload at a flow rate ratio of 1 :1 and a total flow rate of 12 mL / min. The produced formulations were dialysed in 10mM Tris or PBS for a minimum of 4 hours to remove ethanol and NaAC.

[0302] For mRNA as a cargo, the nanoparticle comprises 50.57% ionizable lipid, 40% Cholesterol, 8.43% phospholipid and 1% DMG-PEG and the mRNA: lipid ratio was 1 :22 (Hermes 1). The peptide was K16 and was added to bring the total N / P ratio of the mRNA lipid-peptide nanoparticle to 6.08. The ionizable lipid is preferably ALC-0315, SM-102 or Dlin-MC3-DMA. The phospholipid is preferably DOPE or DSPC. The PEG-lipid is preferably DMG-PEG.

[0303] 57

[0304] 17044091 OMB OMB For closed linear DNA molecule (i.e. hpDNA) as a cargo, the nanoparticle comprises DOTMA, DOTAP, ALC-0315, SM-102 and / or DLin-MC3-DMA as the cationic or ionizable lipid. The molar ratio of lipids was 48.85% cationic / ionizable lipid, 40% Cholesterol, 8.14% DSPC and 3% DMG-PEG (Hermes 3). The mass ratio of hpDNAJipid was 1 :22 and the cationic peptide K16 was added to give a total nitrogen / phosphate ratio (N / P ratio) of 5.8. The nanoparticle may comprise both a cationic and ionizable lipid, preferably at a ratio of 2.35:1. The total molar ratio of the cationic / ionizable component is preferably 48-51 %.

[0305] The components of the nanoparticles used in the Examples are shown in Table 5:

[0306] Table 5. Composition of nanoparticles used in the Examples.

[0307] Analysis of immune responses one week after the second vaccination revealed a significant higher immune response in M20-RNA-Hermes1 treated mice (Figure 7B). The higher level of immune response was less evident after the third vaccination. Tumor challenge on day 52 revealed a significant tumor protection in vaccinated mice with the M20-RNA-Hermes1 (i.e. an mRNA molecule encapsulated in a nanoparticle) performing much better than M20-hpDNA-EP (i.e. a closed linear DNA molecule delivered by electroporation) (Figure 7C). In the second tumor challenge mice which had been vaccinated with M20-RNA-Hermes1 were only partially protected (Figures 7D-E). This suggests a potential issue in the neoantigen-specific long lasting memory T cells. Figure 7 shows that the M20 vaccine formulated as mRNA in nanoparticles (M20-RNA-Hermes 1) induces stronger neoantigen specific CD8+T cell responses compared to M20-hpDNA-EP. A) Experimental design. C57BI / 6 female received three immunizations with the M20 vaccines in a prophylactic setup. One month after the last immunization, mice were inoculated with 3 x 105MC38 tumor cells s.c. in the right flank (n=5) and the tumor size was monitored. Unvaccinated naive mice (n=5) were also challenged with tumour cell line. B) Percentages of IFNy+and TNFa+CD4+and CD8+T cells measured in PBMCs 1 week after the second and last vaccination are shown. PBMCs were restimulated with the peptide pool identified in the ELISpot assay. C) Tumor volumes and survival percentage determined over time are shown. D) & E) Mice immunized with M20-RNA-Hermes1 which remained tumour free were subjected to a second tumour challenge. Graph shows the protective effect of M20-RNA-Hermes1 vaccine. The unpaired t-test was used to make comparisons between two vaccine formulations (B) or untreated group (C, D): **P< 0.01 ; ***P<0.001 .

[0308] 58

[0309] 17044091 OMB OMB Thus, in view of the potential issues with the neoantigen-specific long lasting memory T cells, a heterologous prime-boost system was explored, in which the DNA and RNA based vaccine were formulated in additional formats and delivered in homologous and heterologous prime / boost / boost experiments (Figure 8A). In Figure 8A, “H1-RNA”, denotes an mRNA encapsulated in a Hermes 1 nanoparticle (as per Table 5), “DNA-EP” denotes a closed linear DNA molecule delivered by electroporation, “RNA-EP” denotes an mRNA molecule delivered by electroporation, “H3-DNA” denotes a closed linear DNA molecule encapsulated in a Hermes 3 nanoparticle (as per Table 5) and “LNP-RNA” denotes an mRNA molecule in a formulation known in prior art (as per Table 5, and Schoenmaker et al. 2021) Analysis of immune responses was carried out at different time points (day 7, 21 , 35, and 87). Analysis on day 7 showed that only vaccines in which cargo (here mRNA) was encapsulated in a nanoparticle (in particular H1 , Gr. 2) resulted in a significant immune response already after the first vaccination with a further improvement in an immune response after the subsequent vaccinations (Figure 8B). In contrast, the mRNA molecule delivered by electroporation showed no immune responses at all time points (Figure 8B, RNA-EP, Gr.9). Interestingly, M20- hpDNA-EP or M20-hpDNA-H3 (i.e. the closed linear DNA molecule delivered by either electroporation or in a nanoparticle) used as primer works better than the vaccine with mRNA delivered by electroporation (see Figure 8B, Gr. 5 vs. Gr. 9). A graph representation of the neoantigen-specific CD8+T cells across all groups is depicted in Figure 8C.

[0310] Analysis of the type of memory immune response was conducted after each vaccination. It is interesting to note that activated memory cells (Figures 8D-E) were observed only in M20-hpDNA-EP or M20-RNA-Hermes1 primed mice (Gr. 2-3-4-5) and not in the LNP formulation (Gr.8) of prior art, which served as benchmark.

[0311] Figure 8 shows that the heterologous M20 vaccinations induced strong and durable CD8+T cells as LNPs formulations. A) Experimental design. C57BI / 6 female received three immunizations with the different M20 vaccines and prime / boost / boost protocols. B) C) Percentages of IFNy+and TNFa+CD8+T cells measured in PBMCs. PBMCs were restimulated with the peptide pool identified in the ELISpot assay. D) Percentage of naive, active, effector and central CD8+T cells memory was measured in PBMCs 1 week after each vaccination. PBMCs were restimulated with the peptide pool identified in the ELISpot assay. Graphs show the percentages of neoantigens-specific CD8+T active and effector memory cells, measured after the last vaccination. E) Representative plots of the different treatments acquired by the Northern Lights flow cytometer. The unpaired t-test was used to make comparisons between the vaccine formulations: *P<0.05; **P< 0.01 .

[0312] Finally, to measure the anti-tumor activity associated with the immunogenic protocols described in Figure 8, mice were vaccinated with the same protocol and challenged with MC38 cells (Figure 9A). To correlate the immune responses an ICS analysis was conducted on day 35, 1 week after the last vaccination. Results fully confirmed the results described in the previous experiment with the strongest immune responses in the M20-hpDNA-EP prime and M20-RNA-Hermes1 boost / boost

[0313] 59

[0314] 17044091 OMB OMB (Figure 9B - Gr.6). The analysis of tumor free mice, 3 weeks after the tumor challenge on day 48, showed 50% of tumor free mice and a statistically significant difference with the control group only in the M20-hpDNA-EP prime and M20-RNA-Hermes1 boost / boost treated mice (Figure 9C & D).

[0315] Figure 9 shows that the M20 heterologous prime / boost vaccination resulted in a stronger tumour protection. A) Experimental design. C57BI / 6 female received three immunizations with the M20 formulations in a prophylactic setup. Twelve days after the last immunization, mice were inoculated with 3 x 105MC38 tumor cells s.c. in the right flank (n=5) and the tumor size was monitored. Unvaccinated naive mice (n=5) were also challenged with tumour cell line. B) Percentages of IFNy+ / TNFa+CD4+and CD8+T cells measured in PBMCs 1 week after last vaccination are shown. PBMCs were restimulated with the peptide pool identified in the ELISpot assay. C) Tumor volumes determined over time is shown. D) The graph represents the percentage of tumor-bearing (black) and tumor-free (gray) mice in each experimental group. The unpaired t-test was used to make comparisons between groups: **P< 0.01 , ***P<0.001.

[0316] 3. Conclusion

[0317] The inventors have shown the heterologous prime-boost immunization regimen to be advantageous over a corresponding homologous vaccination regimen; as measured by both the ability to stimulate the immune system to produce IFNy and TNFa and by showing the survival rates of animals suffering from cancer. In addition, the inventors have shown that encapsulation of at least one of the closed linear DNA and the mRNA molecule in a nanoparticle improves the prophylactic and therapeutic effect observed in the context of preventing or treating cancer.

[0318] The data demonstrate that an NCV can be designed, produced, and delivered using closed linear DNA (Figures 1-6), mRNA in a nanoparticle formulation (Figure 7), or various combinations of heterologous prime-boost protocols (Figures 8-9). NCV delivered as a closed linear DNA molecule elicited an immune response that was comparable, if not superior, to that of the plasmid DNA vaccine in the MC38 mouse tumor model, validating the closed linear DNA molecule as a viable alternative delivery platform to plasmid DNA.

[0319] A direct comparison of the closed linear DNA molecule delivered by electroporation and an mRNA molecule delivered in a nanoparticle revealed a significantly stronger immune response triggered by the mRNA molecule comprised in a nanoparticle.

[0320] Tumor challenge data support the effectiveness of a vaccination strategy that primes with closed linear DNA molecule delivered by electroporation, followed by two boosts with an mRNA molecule encapsulated in a nanoparticle. This conclusion is supported by immunological assays, particularly intracellular cytokine staining (ICS) using PBMCs.

[0321] 60

[0322] 17044091 OMB OMB In addition, the heterologous prime-boost approach provides important practical advantages.

[0323] Electroporation of the closed linear DNA molecule enables a faster production process, as the closed linear DNA molecule requires no formulation, a key benefit compared to the longer process needed for the mRNA formulation in a nanoparticle. However, patients primed with a closed linear DNA molecule (via electroporation) can receive a boost 3 weeks later, which corresponds to the time needed to prepare the mRNA-nanoparticle formulation.

[0324] Example 2

[0325] 1. Materials & Methods

[0326] Cell line and mice

[0327] Female 6-7 weeks old C57BL / 6 mice were used for experiments with E40 vaccines (i.e. constructs (either mRNA or closed linear DNA) encoding 40 neoantigens). Animals were housed in the Plaisant animal house and kept in standard condition according to national legislation.

[0328] EO771 cell line was used to induce tumour in C57BL / 6 mice. EO711 cell line is a murine model of Triple negative breast cancer. Master and working cell banks were generated upon receipt, with their third and fourth passages being used for all tumour challenge experiments. Tumour cells were cultured in DMEM medium supplemented with 10% Fetal Bovine Serum (FBS) at 37 °C with 5% CO2. Before experiments, the cell line cultures were subjected to determine for mycoplasma-free status by using a PCR Mycoplasma detection kit (EZ-PCR™ Mycoplasma detection Kit).

[0329] Neoantigens and immunization protocol

[0330] The neoantigens in the E40 vaccine are listed in Table 6. For prophylactic vaccination, C57BL / 6 mice were injected in the tibialis and quadriceps, alternately, bi-weekly or tri-weekly, with different doses of closed linear DNA (hpDNA), and mRNA, formulated as naked material or in liponanoparticles (LNPs) in a volume of 50 pl. Tumour challenge was performed 12 or 30 days after the last immunization.

[0331] In vivo tumour models and evaluation of anti-tumour efficacy

[0332] For tumour inoculation, cells were washed three times with PBS and resuspended in PBS at a density of 0.2x106cells per millilitre. Then, 100 pl was injected subcutaneously into the mammary fat pad of syngeneic recipient mice. Tumour monitoring of tumour was performed by a single operator every three days. Tumour volume was determined using callipers and the formula below. Mice were euthanized when the tumour volume reached 1000 mm3.

[0333] (Lengh x Width2 / ?

[0334] In prophylactic protocol, to highlight the anti-tumour effect induced by the vaccine experiments, mice that rejected tumours after the first challenge, were inoculated with tumour cells on the left flank and tumour growth was monitored over time.

[0335] IFN-y ELIspot assay

[0336] For in vivo immunogenicity assessment of predicted neoantigens, C57BL / 6 mice were injected intramuscularly with 10 pg of E40-hpDNA in a 50 pl volume of Phosphate Buffered Saline (PBS) and electrically stimulated at days indicated in the legend of Figures 10-12. DNA-EP was performed by means of a Cliniporator Device EPS01 and using N-10-4B electrodes (IGEA, Italy) with the following electrical conditions in Electro-Gene-Transfer (EGT) modality: 8 pulses 20 ms each at 110 V, 8 Hz, 120 ms interval. One week after the last vaccination mice were sacrificed and the spleen was explanted for IFNy-Elispot assay according to the manufacturer’s instructions (Mouse IFN-gamma ELISpotBASIC ALP, Mabtech). Briefly, standard 96-well plates (Millipore) were coated with anti-mouse IFNy antibody diluted to 15 pg / ml in sterile PBS and blocked with RPMI-1640 medium with 10% FBS. Splenocytes were plated at 3 x 105, in duplicate, with 20-mer peptide coding the sequence of E40 vaccine (as shown in Table 7) at the final concentration of 1 pg / ml. After overnight stimulation at 37 °C, plates were washed and incubated with biotinylated anti-mouse IFNy antibody, washed, and incubated for 1 h at room temperature with streptavidin-AP conjugated antibody. After washing, 50 pl / well of the substrate (NBT / BCIP-1 step solution, Pierce) was added to measure spot development. Finally, the plates were thoroughly washed with distilled water to stop the reaction and were allowed to air-dry completely. Spot forming colonies (SFC) were counted using an automated ELISPOT reader (Aelvis ELIspot reader, A.EL.VIS Gmbh).

[0337] Immune responses

[0338] The neoantigen-specific immune responses were determined in PBMCs of the mice by using intracellular cytokine staining (ICS) performed by flow cytometry (FC). Briefly, blood was incubated for 10 min at room temperature in ACK (Ammonium-Chloride- Potassium) Lysing Buffer and then washed in RPMI-1640 medium supplemented with 10% FBS. For FC analysis of IFNy producing cells, PBMCs were cultured in 96-well plates and stimulated for 12-16 h in 10% FBS-supplemented RPMI-1640 with the single E40 neoantigen peptide at the final concentration of 5 pg / ml, DMSO or PMA-ionomycin at 10pg / ml were used as internal negative and positive control of the assay, respectively. The E40 pool contain immunogenic peptides identified in the ELISpot assay and are listed in Table 8. For FC analysis, dead cells were excluded by using Fixable Viability Stain. Cells were incubated with the anti-Fcy receptor (2.4G2) followed by staining with the following antibodies: CD45 (30-F11), CD3 (142-2C11), CD4 (RM4-5), CD8 (53-6.7), IFNy (XMG1.2), and TNFa (MP6-XT22). The stained samples were acquired through a Northen lights flow cytometer (Cytek), and the data were analyzed using Cytek software. Cytokine expression in the presence of only DMSO (with no peptides) was considered background and subtracted from the values measured with stimuli.

[0339] Statistics

[0340] GraphPad Prism software (v8.0.2) was used to perform all statistical analyses. Significance was determined using Mann-Whitney test. *P< 0.05 was considered statistically significant. **P< 0.01 , ***P < 0.001 , ****P<0.0001 . All data were presented as means ± SD.

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[0342] 17044091 OMB OMB

[0343] Table 6. List of 40 neoantigens in E40 constructs.

[0344] Table 7. List of 20-mer peptides included in E40-pool used for in vitro lymphocyte stimulation.

[0345] Lipid nanoparticles

[0346] The nanoparticles were prepared as in Example 1 . In Example 2, only the mRNA was encapsulated. For this, the composition of the nanoparticles was shown in Table 5 - see Hermes 1 .

[0347] 2. Results

[0348] Neoantigen identification and immunogenicity evaluation for mouse Triple Negative breast cancer model

[0349] The approach described in Example 1 was further optimised to develop a vaccine with 40 neoantigens. The intention was for the immunogenic sequences to be capable of effectively activating CD4+and / or CD8+T cell responses. To this aim neoantigens were selected using the algorithms implemented as in Example 1 (see also Example 2, Materials & Methods), based on three main criteria: predicted binding affinity to MHC class I (HBI), binding to MHC class II (HBII), and gene expression levels (MRE). For each sample, two separate rankings were generated — one for predicted CD4+T cell activation and another for CD8+T cell activation. Once individual rankings were established, an intersection analysis was performed across all samples to identify 140 neoantigens commonly shared among the EO771 cell lines (i.e. a syngeneic mouse mammary cancer cell line). This step was essential to identify the most relevant candidates, specifically those neoantigens recurrent across multiple samples, which are more likely to represent robust targets for therapeutic strategies. Finally, the mean ranking for each neoantigen was calculated across all samples, separately for CD4 and CD8 responses. Based on the resulting average rankings, we defined the composition of the E40 vaccine, which includes: the top 20 neoantigens for CD4+T cell responses and the top 20 neoantigens for CD8+T cell responses. Neoantigens present in E40 construct are listed in Table 6.

[0350] The predicted neoantigens immunogenicity was validated in syngeneic murine model: EO771 tumour implanted in mammary fat pad of C57 / BI6 mice. Similarly to M20 and C20 (Example 1), the specificity of the immunological response was measured by ELISpot assay conducted using single 20-mer peptides spanning full length E40 vaccine as the stimulant. IFN-y ELISpot analysis on splenocytes gave the results depicted in Figure 10.

[0351] Figure 10 illustrates the identification of immunogenic neoantigens induced by E40 (i.e. a closed linear DNA molecule delivered by electroporation or mRNA encapsulated in a nanoparticle (termed Hermes))

[0352] 64

[0353] 17044091 OMB OMB vaccination. A) Experimental design: C57BI / 6 female mice were immunized by electroporation (EP) on days 0, 21 and 42 with 10 pg of a closed linear DNA molecule construct encoding for the 40 neoantigens from EO771 model. 1 -week after the last vaccination mice were sacrificed and the spleen was explanted for IFNy ELISpot assay. B) Spot forming colonies (SFC) secreting IFNy upon stimulation with single 20- mer peptide coding sequence of E40 vaccine. Graph shows peptides resulting positive. C) Experimental design: C57BI / 6 female mice were vaccinated on days 0 and 14 with 10 pg of mRNA molecule construct encoding for the 40 neoantigens from EO771 model. 1-week after the last vaccination mice were sacrificed and the spleen was explanted for IFNy ELISpot assay. D) Spot forming colonies (SFC) secreting IFNy upon stimulation with single 20-mer peptide coding sequence of E40 vaccine. Graph shows peptides resulting positive. E-F) illustrates ICS analysis by flow cytometry of neoantigens identified in the Figure 10. CD4+and CD8+T cells were identified by IFNy ICS analysis. The unpaired t-test was used to make comparisons between groups: *P<0.05, **P< 0.01 , ****P<0.0001 . Table 8 summarizes the peptide code, the sequence and the related gene. The corresponding 28-mer sequences are shown in Table 7.

[0354] Table 8. Summary of the peptide code, the sequence and the related gene as expressed by the E40 construct for the data shown in Figure 10.

[0355] In particular, out of the 44 peptides tested, 11 elicited a positive response following DNA vaccination, and 12 following RNA vaccination. Thus, with the exception of gene Bhlhe40, the peptide-specific responses overlapped between the two vaccine types. ICS analysis identified peptide #5 (corresponding to Ftsj3 gene) as the most immunogenic CD8 epitope, and peptide #40 (corresponding to Plekhg3 gene) as the most immunogenic CD4 epitope in both the E40-hpDNA and E40-mRNA-lipid nanoparticle-based (Hermes) vaccines.

[0356] Antitumor efficacy evaluation of breast cancer neoantigen vaccines

[0357] To investigate the anti-tumor activity of neoantigen peptides, mice were vaccinated in a prophylactic protocol, with 10 pg of E40-hpDNA-EP or E40-mRNA-Hermes, and 1 month or 12 days after the last

[0358] 65

[0359] 17044091 OMB OMB vaccination were injected with EO771 cells as describe in material and methods section. Results showed a relevant anti-tumor effect (Figure 11).

[0360] Figure 11 shows that E40 vaccination (i.e. a closed linear DNA molecule delivered by electroporation or a mRNA molecule encapsulated in a lipid nanoparticle (termed Hermes)) induce a significant tumour protection. A) Experimental design. C57BI / 6 female mice received three immunizations with electroporation (EP) in a prophylactic setup. One month after the last immunization, mice were inoculated with 0.2 x 106EO771 tumour cells in mammary fat pad injection (n=10) and the tumour size was monitored. Unvaccinated naive mice (n=5) were also challenged with tumour cell line. B) Tumour volumes and survival curves determined over time are shown. C) Experimental design. C57BI / 6 female mice received two immunizations with E40-mRNA-Hermes in a prophylactic setup. 13 days after the last immunization, mice were inoculated with 0.2 x 106EO771 tumour cells in mammary fat pad injection (n=10) and the tumour size was monitored. Unvaccinated naive mice (n=5) were also challenged with tumour cell line. D) Tumour volumes and survival curves determined over time are shown. The unpaired t-test was used to make comparisons: *P<0.05, **P< 0.01 .

[0361] Evidence of vaccine-induced tumour immunity is supported by the failure of rechallenge engraftment in mice that have previously rejected tumours, alongside successful engraftment of the same tumour in naive animals as a control (Figure 12).

[0362] Figure 12 illustrates the long-time protective effect of E40-hpDNA-EP vaccine in a prophylactic vaccination protocol. A) Experimental design. C57BI / 6 female mice received three immunizations with electroporation (EP) in a prophylactic setup. Two months after the first challenge, mice which remained tumour free after first challenge were subjected to a second tumour challenge (0.2 x 106EO771 tumour cells in mammary fat pad injection (on the left) (n=10)). Unvaccinated naive mice (n=5) were also challenged with tumour cell line. B) Tumour volumes and survival curves determined over time are shown. The unpaired t-test was used to make comparisons: **P< 0.01 ; ***P<0.001 .

[0363] 3. Conclusions

[0364] The inventors have successfully generated two different types of cancer vaccines - one DNA-based, and one RNA-based, each encoding for 40 different neoantigens. The generated vaccines triggered both CD4+ and CD8+ T cells activity due to the affinity of some of the neoantigens for MHC class I (HBI) and the remaining neoantigens for MHC class II (HBII). Some of the neoantigens encoded by the vaccine may show affinity for both the MHC class I and MHC class II.

[0365] The inventors identified neoantigens which are particularly effective in triggering immune response in the context of triple negative breast cancer model (Table 8). These neoantigens were effective when encoded by both the DNA-based and RNA-based vaccines.

[0366] 66

[0367] 17044091 OMB OMB The inventors have validated the generated vaccines in preventing the formation of a breast cancer in mice model.

[0368] The present vaccine technology is associated with a significant advantage as it allows to account for tumour heterogenicity. In other words, not only the inventors identified a high number of neoantigens that are effective in triggering immune response in a breast cancer mice model, the inventors have also allowed for genetic variety of different cancer cells. This allows for a universal use of the technology, potentially by administering the same vaccine (encoding ~40 neoantigens) to any patient with a given cancer type or predicted to have a given cancer type.

[0369] Example 3

[0370] The lipid nanoparticles used in Examples 1 and 2 were analysed for their biophysical characteristics. Figure 13 shows biophysical characteristics of lipid nanoparticle formulations. A) Size, B) polydispersity index (PDI) and C) encapsulation efficiency (EE%) of Hermes nanoparticles or control LNP of prior art encapsulating mRNA or closed linear DNA encoding 20 neoantigens against the MC38 tumour model (M20) or 40 neoantigens against the EO771 tumour model (E40). n = 3.

[0371] The critical quality attributes of all nanoparticles prepared for these experiments met the acceptance criteria. All formulations had a diameter under 200 nm with low PDI values under 0.2, indicating a uniform, polydisperse population, as measured by dynamic light scattering (Malvern Zetasizer). Encapsulation efficiency, measured by ribogreen or picogreen (mRNA or hpDNA, respectively, ThermoFisher Scientific), demonstrated that -95% of payload was encapsulated in the nanoparticle.

Claims

CLAIMS1 . A combination comprising: a. a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens; and b. a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens; wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

2. The combination of claim 1 , wherein the mRNA molecule is comprised in a nanoparticle, and optionally wherein the closed linear DNA molecule is delivered to a subject by electroporation.

3. The combination of claim 1 or claim 2, wherein the closed linear DNA molecule encodes at least three, at least four, at least five, at least 10, at least 20, at least 30, or at least 40 different neoantigens and / or the mRNA molecule encodes at least three, at least four, at least five, at least 10, at least 20, at least 30, or at least 40 different neoantigens, optionally wherein the closed linear DNA and the mRNA molecule encode at least 1 , at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, or at least 40 of the same neoantigens.

4. The combination of any one of claims 1-3, wherein the nanoparticle comprises: a. a lipid component, wherein the lipid component is one or more ionizable lipids and / or one or more cationic lipids; b. a phospholipid; c. a steroid lipid; and d. a cationic polymer, optionally wherein the cationic polymer is a polycationic peptide and / or a nucleic acid-binding cationic component.

5. The combination of any one of claims 1 -4, wherein the first vaccine composition is delivered to a subject at a first time point, and the second vaccine composition is delivered to the subject at a second time point, optionally wherein the second time point is at least 14 days after the first time point.

6. The combination of any one of claims 1 -5, wherein the first vaccine composition is delivered to a subject at a first time point by electroporation, and the second vaccine composition, which is comprised in a nanoparticle, is delivered to the subject at a second time point.

7. The combination of any one of claims 1-6, wherein the combination comprises a third vaccine composition, wherein the third vaccine composition comprises a mRNA molecule encoding at least two neoantigens, optionally wherein the third vaccine composition is delivered to the subject at a third time point, and optionally wherein the third time point is at least 14 days after the second time point.

8. The combination of any one of claims 1-7, wherein the combination further comprises an immune checkpoint inhibitor.

9. A combination or kit-of-parts comprising(a) a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens; and (b) a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens, for simultaneous, separate or sequential use in treating or preventing cancer in a subject, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

10. A first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens for use in treating or preventing cancer, wherein the first vaccine composition is administered to a subject simultaneously, separately or sequentially with a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.11 . A second vaccine composition comprising a mRNA molecule encoding at least two neoantigens for use in treating or preventing cancer, wherein the second vaccine composition is administered to a subject simultaneously, separately or sequentially with a first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

12. A first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens for use in a heterologous prime-boost immunization regimen in a subject, wherein the first vaccine composition is administered at least once to the subject, and wherein a second vaccine composition, comprising an mRNA molecule encoding at least two neoantigens is separately administered to the subject following at least one administration of the first vaccine, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

13. A first vaccine composition comprising a closed linear DNA molecule encoding at least two neoantigens for use in treating or preventing cancer in a subject, wherein the first vaccinecomposition is administered at least once to the subject, and wherein a second vaccine composition comprising an mRNA molecule encoding at least two neoantigens is separately administered to the subject following at least one administration of the first vaccine, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

14. A second vaccine composition comprising an mRNA molecule encoding at least two neoantigens for use in a heterologous prime-boost immunization regimen in a subject, wherein the second vaccine composition is administered to the subject following at least one administration of a first vaccine composition, and wherein the first vaccine composition comprises a closed linear DNA molecule encoding at least two neoantigens, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

15. A second vaccine composition comprising an mRNA molecule encoding at least two neoantigens for use in treating or preventing cancer in a subject, wherein the second vaccine composition is administered to the subject following at least one administration of a first vaccine composition, and wherein the first vaccine composition comprises a closed linear DNA molecule encoding at least two neoantigens, wherein the closed linear DNA molecule and / or the mRNA molecule are comprised in a nanoparticle.

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